Protein Blotting Methods

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en-us LUSPPSESH Protein Blotting Methods Protein Blotting Methods /webroot/web/html/lsr/solutions/technologies/western_blotting <p>The initial step in any blotting experiment is the transfer of proteins from a solution or gel and immobilization of those proteins on a solid membrane support. Immobilization of proteins on a membrane makes the proteins accessible to probes for specific proteins and enables quantitative detection. This section describes the principle of electrophoretic transfer of proteins, and details tank blotting, semi-dry blotting, and microfiltration or dot-blotting methods.</p> <p><strong>Related Topics</strong>: <a href="/evportal/destination/solutions?catID=LUSPQXC4S">Protein Blotting Equipment</a>, <a href="/evportal/destination/solutions?catID=LUSPSJ8UU">Membranes and Blotting Paper</a>, <a href="/evportal/destination/solutions?catID=LUSPTIMNI">Transfer Conditions</a>, <a href="/evportal/destination/solutions?catID=LUSPULKSY">Protein Blotting Detection and Imaging</a>, <a href="/evportal/destination/solutions?catID=LUSQA88UU">Transfer Buffers</a>, and <a href="/evportal/destination/solutions?catID=LUSQON470">Imaging and Analysis</a>.</p> Protein Blotting Methods <p>The two most common methods for protein transfer are:</p> <ul> <li><strong>Electrophoretic transfer</strong> &mdash; proteins separated in gels (for example, following polyacrylamide gel electrophoresis, or PAGE), are transferred onto membranes by electrophoretic transfer</li> <li><strong>Microfiltration (dot blotting)</strong> &mdash; proteins in solution are bound to membranes by vacuum driven microfiltration. This rapid approach is useful for determining working conditions for a new blotting assay or any other situation where the resolving power of gel electrophoresis is not needed</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Protein Transfer Methods <p><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/pet21_protein_transfer_methods.gif" alt="Protein Transfer Methods" /></p> <p class="caption"><strong>Protein transfer methods.</strong></p> <div class="top"><a href="#helptop">Back to Top</a></div> Electrophoretic Transfer <p><strong>Electrophoretic Transfer Setup</strong><br /> In electrophoretic transfer, an electric field is used to elute proteins from gels and transfer them to membranes. Electrophoretic transfer is the most widely used blotting method because of its speed and precision in replicating the pattern of separated proteins from a gel to a membrane.</p> <p>In an electrophoretic transfer, the membrane and protein-containing gel are placed together, with filter paper between two electrodes.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/pet21_gel_membrane_setup.gif" alt="Gel and membrane setup for electrophoretic transfer." width="385px" height="282px" /></p> <p class="caption"><strong>Gel and membrane setup for electrophoretic transfer.</strong></p> <p><strong>Electrophoretic Transfer Principle</strong></p> <p>Proteins migrate to the membrane following a current (I) that is generated by applying a voltage (V) across the electrodes* following Ohm's law:</p> <p style="margin-left:25px; font-weight: bold; font-size: larger;">V = I x R</p> <p>R is the resistance generated by the materials placed between the electrodes (that is, the transfer buffer, gel, membrane, and filter papers).</p> <p>The electric field strength (E, measured in V/cm) that is generated between the electrodes is the driving force for transfer. Both the applied voltage and the distance between the electrodes then play a major role in governing the rate of elution of the proteins form the gel. A number of other factors, including the size, shape, and charge of the protein*, the pH, viscosity, and ionic strength of the transfer buffer, and the composition of the gel, also influence the elution of particular proteins from gels.</p> <p>There are practical limits on field strength, however, due to the production of heat during transfer. The heat generated (Joule heating) is proportional to the power consumed by the electrical elements (P), which is equal to the product of the current (I) and voltage (V):</p> <p style="margin-left:25px; font-weight: bold; font-size: larger;">P = I x V = I<sup>2</sup> x R</p> <p>Joule heating increases temperature and decreases resistance of the transfer buffer. Such changes in resistance may lead to inconsistent field strength and transfer, or may cause the transfer buffer to lose its buffering capacity. In addition, excessive heat may cause the gel to deteriorate and stick to the membrane. The major limitation of any electrophoretic transfer method is the ability of the chamber to dissipate heat.</p> <p class="caption">* Proteins denatured with sodium dodecyl sulfate (SDS) carry a net negative charge and migrate toward the anode.</p> <p><strong>Electrophoretic Transfer Systems</strong><br /> There are two main types of electrophoretic blotting apparatus and transfer procedures (see <a href="#comparison">table below</a>):</p> <ul> <li><strong>Tank transfer systems</strong> &mdash; gels and membranes are submerged in transfer buffer in tanks; these systems are useful for most routine protein work, for efficient and quantitative protein transfers, and for transfers of proteins of all sizes. Tank transfer systems offer the most flexibility in choosing voltage settings, blotting times, and cooling options</li> <li><strong>Semi-dry systems</strong> &mdash; gels and membranes are sandwiched between buffer-saturated filter papers that are in direct contact with plate electrodes; these systems are typically easier to set up than tank systems and are useful when high-throughput is necessary and extended transfer times are not required, or when discontinuous buffer systems are used. Active cooling options are limited with semi-dry blotting</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Tank Blotting <p>In tank blotting systems, the gel and membrane sandwich is entirely submerged under transfer buffer within a buffer tank. A non-conducting cassette holds the membrane in close contact with the gel and the cassette assembly is placed in the tank between the electrodes, transverse to the electrical field, and submerged under conducting transfer buffer (Burnette 1981, Gershoni et al. 1985, Towbin et al. 1979). Although the large volumes of buffer in the tank dissipate the heat generated during transfer and provide the conducting capacity for extended transfer conditions, additional cooling mechanisms are offered by the various <a href="/evportal/destination/solutions?catID=LUSPQXC4S">tank blotting systems</a>.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Semi-Dry Blotting <p>In a semi-dry transfer, the gel and membrane are sandwiched between two stacks of filter paper that are in direct contact with plate electrodes (Bjerrum and Schafer-Nielsen 1986, Kyhse-Andersen 1984, Tovey and Baldo 1987). The term semi-dry refers to the limited amount of buffer, which is confined to the two stacks of filter paper.</p> <p>In semi-dry systems, the distance between the electrodes is limited only by the thickness of the gel and membrane sandwich. As a result, high electric field strengths and high-intensity blotting conditions are achieved. Under semi-dry conditions, some small proteins may be driven through the membrane in response to the high field strengths. Moreover, because low buffer capacity limits run times, some large proteins may be poorly transferred. Use of a discontinuous buffer system, that utilizes different buffer formulations on the anode and the cathode side of the transfer sandwich (see <a href="/evportal/destination/solutions?catID=LUSQA88UU">Transfer Buffers</a>) may enhance semi-dry transfer of high molecular weight proteins (&gt;80 kD). As semi-dry transfers require considerably less buffer and are easier to set up than the tank method, laboratories performing large numbers of blots often favor them.</p> <p>Novel buffer and material formulations have been developed that can be used with higher electric field strengths than those used in typical semi-dry blotting. These conditions yield complete and extremely rapid transfer, with some systems completing transfer in 3&ndash;10 min. Such rapid blotting systems do not incorporate external cooling mechanisms, so the high power dissipation may generate more heat than other techniques. Rapid blotting systems are intended for extremely rapid transfers where heat-induced protein denaturation will not affect downstream applications.</p> <p>Choose the right <a href="http://www.bio-rad.com/evportal/destination/product?catID=5290044b-3d9b-4a5e-987b-9c3214086bd4">semi-dry or rapid transfer system</a> based on the demands of your application.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Microfiltration (Dot Blotting) <p>Simple bulk transfer of proteins that are in solution may be achieved by manual application (dotting) to a membrane from a pipet or syringe, or by vacuum-assisted microfiltration. Manual dot-blotting with a pipet or syringe is generally used for small sample volumes. Microfiltration devices, on the other hand, enable application of larger volumes, multiple assays with different probes, and quick, reproducible screening of a large number of samples. Microfiltration facilitates the determination of working conditions for a new blotting assay and is a convenient method in any other situation where the resolving power of gel electrophoresis is not needed.</p> <p><a name="comparison"></a></p> <p><strong>Comparison of electrophoretic protein transfer systems.</strong></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td style="width: 149px;">&nbsp;</td> <td style="width: 149px;" align="center"><strong>Tank Blotting</strong></td> <td style="width: 298px;" colspan="2" align="center"><strong>Semi-Dry Blotting</strong></td> </tr> <tr> <td>&nbsp;</td> <td>&nbsp;</td> <td style="width: 149px;" align="center"><strong>Traditional</strong></td> <td style="width: 149px;" align="center"><strong>Rapid</strong></td> </tr> <tr> <td style="border-top:1px solid #000" valign="top"><strong>Transfer Time</strong></td> <td style="border-top:1px solid #000" valign="top">30 min&ndash;overnight</td> <td style="border-top:1px solid #000" valign="top">15&ndash;60 min</td> <td style="border-top:1px solid #000" valign="top">3&ndash;10 min</td> </tr> <tr class="pd_colorbackground"> <td valign="top"><strong>Handling convenience</strong></td> <td valign="top">Manual assembly of transfer components</td> <td valign="top">Manual assembly of transfer components</td> <td valign="top">Prepackaged, presaturated components</td> </tr> <tr> <td valign="top"><strong>Transfer parameters</strong></td> <td valign="top">Widest range of power settings and transfer times</td> <td valign="top">Power and transfer time limited due to lack of cooling options</td> <td valign="top">Preinstalled, customizable programs for transfer of most proteins, user-programmable settings for traditional semi-dry techniques</td> </tr> <tr class="pd_colorbackground"> <td valign="top"><strong>Molecular weight range</strong></td> <td valign="top">Broad range</td> <td valign="top">Best for 30&ndash;120 kD</td> <td valign="top">Broad range</td> </tr> <tr> <td valign="top"><strong>Temperature control</strong></td> <td valign="top">Cooling with ice pack or refrigerated water recirculator</td> <td valign="top">None</td> <td valign="top">None</td> </tr> <tr class="pd_colorbackground"> <td style="border-bottomx:1px solid #000" valign="top"><strong>Buffer requirement</strong></td> <td style="border-bottomx:1px solid #000" valign="top">1&ndash;12 L, system-dependent</td> <td style="border-bottomx:1px solid #000" valign="top">250 ml per blot</td> <td style="border-bottomx:1px solid #000" valign="top">No additional buffer required</td> </tr> </tbody> </table> <div class="top"><a href="#helptop">Back to Top</a></div> References <p>Bjerrum OJ and Schafer-Nielsen C (1986). Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In Electrophoresis '86: Proceedings of the Fifth Meeting of the International Electrophoresis Society, M.J. Dunn, ed. (Weinheim, Germany: Wiley-VCH Verlag GmbH), pp. 315&ndash;327.</p> <p>Burnette WN (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195&ndash;203.</p> <p>Gershoni JM (1985). Protein blotting: developments and perspectives. Trends Biochem Sci 10, 103&ndash;106.</p> <p>Kyhse-Andersen J (1984). Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10, 203&ndash;209.</p> <p>Tovey ER and Baldo BA (1987). Comparison of semi-dry and conventional tank-buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes. Electrophoresis 8, 384&ndash;387.</p> <p>Towbin H et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350&ndash;4354.</p> <div class="top"><a href="#helptop">Back to Top</a></div> 2895 Protein Blotting Guide, Ver C 2895 /webroot/web/pdf/lsr/literature/Bulletin_2895.pdf Literature PDF Other /webroot/web/images/general/icons/icon_pdf.gif Protein Blotting Guide No Protein Blotting Guide, Ver C 2895 Bulletin 2895, horseradish peroxidase (HRP), enzyme-antibody complex, alkaline phosphatase (AP), nitroblue tetrazolium (NBT) tablets, BCIP, secondary antibody probe, amido black, streptavidin-biotin, anionic dye, total protein blotting stain, nitrocellulose, supported nitrocellulose or polyvinylidene difluoride (PVDF) or low fluorescence LF PVDF membranes, antibody, immunoglobulin (IgG), antigen, assay, avidin, biotin, background, non-specific noise, Bjerrum Schafer-Nielsen buffer, blocking reagent, Western blotting, BLOTTO, chemiluminescence, colloidal gold, color development reagent, colorimetric detection, chemiluminescent detection, enzyme-antibody conjugate, Coomassie Blue stain, diaminobenzidine (DAB), dot blot, Dunn buffer, electrophoretic blotting, foam pads, filter paper, gelatin, high-intensity transfer, immunoassay, immunoblotting, immunodetection, ligand, membrane, membrane/filter paper sandwiches, microfiltration blotting, multiplexing, multiscreen apparatus, native PAGE, polyacrylamide gel electrophoresis, NHS-biotin, non-enzymatic probe, nonenzymatic probe, non-fat dry milk, non-specific binding, phycobiliprotein, power supply, primary antibody, prestained standards, molecular weight markers or ladders, Protein A, Protein G, rapid semi-dry blotting, SDS-PAGE, sodium dodecyl sulfate, sodium dodecyl sulphate, signal-to-noise ratio, signal to noise ratio, Stain-free technology, CCD imager, charge-coupled device, StrepTactin, Strep-tag sequence, substrate, super cooling coil, tank blotting, Towbin buffer, Tween 20 2032 Western Blotting Detection Reagents Brochure, Rev F 2032 /webroot/web/pdf/lsr/literature/Bulletin_2032F.pdf Literature PDF Product_Information_Sheets /webroot/web/images/general/icons/icon_pdf.gif Western Blotting Detection Reagents Brochure No Western Blotting Detection Reagents Brochure, Rev F 2032 western, blotting, blot, protein, reagent, specific antigen, horseradish peroxidase, alkaline phosphatase, primary, secondary, antibody, conjugate, conjugates, signal, total protein, block nonspecific sites, incubate, fluorescent stain, anionic dye, colloidal gold, chemiluminescence, color development, selection guide, immune-star, westernc, chemiluminescent, hrp, ap, amplified opti-4cn, dab, immun-blot, enzyme-conjugated antibodies, substrate, luminal, cdp-star, nitrocellulose, pvdf membrane blots, ccd imager, quantity one software, 170-5040, 170-5041, 170-5043, 170-5042, 170-5044, 170-5045, 170-5047, 170-5046. 170-5010, 170-5011, 170-5012, 170-5018, 170-5070, 1705040, 1705041, 1705043, 1705042, 1705044, 1705045, 1705047, 1705046, 1705010, 1705011, 1705012, 1705018, 1705070, bio-safe coomassie blue, uv, sypro ruby, 170-6527, 170-3127, 161-0786, 1706527, 1703127, 1610786, colorimetric, film-based, film, bcip/nbt, bcip, nbt, 4cn, premixed, sensitivity, background, 170-6463, 170-6464, 170-6465, 170-6460, 170-6461, 170-6462, 170-8237, 170-8239, 170-8240, 170-8235, 170-8238, 170-6432, 170-6431, 170-6534, 170-6539, 170-6532, 170-6535, 170-6518, 170-6520, 170-6521, 170-6515, 170-6516, 172-1050, 170-6533, 170-6528, 170-3554, 170-6522, 170-6425, powdered, liquid, standards, precision plus protein, strep-tag, strep-tagged, streptactin, on-blot, molecular weight estimation, transfer efficiency, unstained, dual color, xtra, kaleidoscope, all blue, biotinylated, prestained sds-page, band pattern, monitoring, low range, high, broad, avidin, 161-0376, 161-0385, 161-0363, 161-0374, 161-0394, 161-0377, 161-0375, 161-0395, 161-0373, 161-0393, 161-0305, 161-0309, 161-0318, 161-0324, 161-0325, 161-0311, 161-0306, 161-0319, 161-0312, 161-0313, 161-0307, 161-0308, 161-0321, 161-0322, 161-0380, 161-0382, 1610376, 1610385, 1610363, 1610374, 1610394, 1610377, 1610375, 1610395, 1610373, 1610393, 1610305, 1610309, 1610318, 1610324, 1610325, 1610311, 1610306, 1610319, 1610312, 1610313, 1610307, 1610308, 1610321, 1610322, 1706528, 1706533, 1610380, 1610382, 170-5015, CDPstar, 1705015 Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Mini Format 1D-Electrophoresis Systems/Mini-PROTEAN Precast Gels/Mini-PROTEAN TGX Stain-Free Precast Gels ->MTS::L0U6RK15##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Semi-Dry Blotting Systems/Trans-Blot Turbo Transfer System ->MTS::LGOQBW15##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Western Blotting Membranes and Filter Paper/Immun-Blot PVDF (for Western Blotting) ->MT::9f95f7dc-8ad2-4923-8eb4-be822b64e2f4##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Immunodetection Reagents and Kits/Immun-Star HRP Chemiluminescence Kits ->MT::e08909eb-3369-42ec-a585-df189004268f## Life Science Research/Solutions/Technologies/Protein Electrophoresis ->MTS::LUSOVO47B##Life Science Research/Solutions/Technologies/Protein Electrophoresis/Protein Detection and Analysis ->MTS::LUSPLU15## Life Science Research/Solutions/Applications/Protein Separation and Analysis/Fast Separation Transfer and Analysis ->MTS::LUSOUMFCN## Eddie C Protein Blotting Methods 12/08/11 10:59 AM 12/08/21 11:02 AM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/Western_Blotting N 0 Introduction to Western Blotting /en-us/applications-technologies/applications-technologies/protein-blotting-methods?ID=LUSPPAKG4

The initial step in any blotting experiment is the transfer of proteins from a solution or gel and immobilization of those proteins on a solid membrane support. Immobilization of proteins on a membrane makes the proteins accessible to probes for specific proteins and enables quantitative detection. This section describes the principle of electrophoretic transfer of proteins, and details tank blotting, semi-dry blotting, and microfiltration or dot-blotting methods.

Related Topics: Protein Blotting Equipment, Membranes and Blotting Paper, Transfer Conditions, Protein Blotting Detection and Imaging, Transfer Buffers, and Imaging and Analysis.

 

Protein Blotting Methods

The two most common methods for protein transfer are:

  • Electrophoretic transfer — proteins separated in gels (for example, following polyacrylamide gel electrophoresis, or PAGE), are transferred onto membranes by electrophoretic transfer
  • Microfiltration (dot blotting) — proteins in solution are bound to membranes by vacuum driven microfiltration. This rapid approach is useful for determining working conditions for a new blotting assay or any other situation where the resolving power of gel electrophoresis is not needed
 

Protein Transfer Methods

Protein Transfer Methods

Protein transfer methods.

 

Electrophoretic Transfer

Electrophoretic Transfer Setup
In electrophoretic transfer, an electric field is used to elute proteins from gels and transfer them to membranes. Electrophoretic transfer is the most widely used blotting method because of its speed and precision in replicating the pattern of separated proteins from a gel to a membrane.

In an electrophoretic transfer, the membrane and protein-containing gel are placed together, with filter paper between two electrodes.

Gel and membrane setup for electrophoretic transfer.

Gel and membrane setup for electrophoretic transfer.

Electrophoretic Transfer Principle

Proteins migrate to the membrane following a current (I) that is generated by applying a voltage (V) across the electrodes* following Ohm's law:

V = I x R

R is the resistance generated by the materials placed between the electrodes (that is, the transfer buffer, gel, membrane, and filter papers).

The electric field strength (E, measured in V/cm) that is generated between the electrodes is the driving force for transfer. Both the applied voltage and the distance between the electrodes then play a major role in governing the rate of elution of the proteins form the gel. A number of other factors, including the size, shape, and charge of the protein*, the pH, viscosity, and ionic strength of the transfer buffer, and the composition of the gel, also influence the elution of particular proteins from gels.

There are practical limits on field strength, however, due to the production of heat during transfer. The heat generated (Joule heating) is proportional to the power consumed by the electrical elements (P), which is equal to the product of the current (I) and voltage (V):

P = I x V = I2 x R

Joule heating increases temperature and decreases resistance of the transfer buffer. Such changes in resistance may lead to inconsistent field strength and transfer, or may cause the transfer buffer to lose its buffering capacity. In addition, excessive heat may cause the gel to deteriorate and stick to the membrane. The major limitation of any electrophoretic transfer method is the ability of the chamber to dissipate heat.

* Proteins denatured with sodium dodecyl sulfate (SDS) carry a net negative charge and migrate toward the anode.

Electrophoretic Transfer Systems
There are two main types of electrophoretic blotting apparatus and transfer procedures (see table below):

  • Tank transfer systems — gels and membranes are submerged in transfer buffer in tanks; these systems are useful for most routine protein work, for efficient and quantitative protein transfers, and for transfers of proteins of all sizes. Tank transfer systems offer the most flexibility in choosing voltage settings, blotting times, and cooling options
  • Semi-dry systems — gels and membranes are sandwiched between buffer-saturated filter papers that are in direct contact with plate electrodes; these systems are typically easier to set up than tank systems and are useful when high-throughput is necessary and extended transfer times are not required, or when discontinuous buffer systems are used. Active cooling options are limited with semi-dry blotting
 

Tank Blotting

In tank blotting systems, the gel and membrane sandwich is entirely submerged under transfer buffer within a buffer tank. A non-conducting cassette holds the membrane in close contact with the gel and the cassette assembly is placed in the tank between the electrodes, transverse to the electrical field, and submerged under conducting transfer buffer (Burnette 1981, Gershoni et al. 1985, Towbin et al. 1979). Although the large volumes of buffer in the tank dissipate the heat generated during transfer and provide the conducting capacity for extended transfer conditions, additional cooling mechanisms are offered by the various tank blotting systems.

 

Semi-Dry Blotting

In a semi-dry transfer, the gel and membrane are sandwiched between two stacks of filter paper that are in direct contact with plate electrodes (Bjerrum and Schafer-Nielsen 1986, Kyhse-Andersen 1984, Tovey and Baldo 1987). The term semi-dry refers to the limited amount of buffer, which is confined to the two stacks of filter paper.

In semi-dry systems, the distance between the electrodes is limited only by the thickness of the gel and membrane sandwich. As a result, high electric field strengths and high-intensity blotting conditions are achieved. Under semi-dry conditions, some small proteins may be driven through the membrane in response to the high field strengths. Moreover, because low buffer capacity limits run times, some large proteins may be poorly transferred. Use of a discontinuous buffer system, that utilizes different buffer formulations on the anode and the cathode side of the transfer sandwich (see Transfer Buffers) may enhance semi-dry transfer of high molecular weight proteins (>80 kD). As semi-dry transfers require considerably less buffer and are easier to set up than the tank method, laboratories performing large numbers of blots often favor them.

Novel buffer and material formulations have been developed that can be used with higher electric field strengths than those used in typical semi-dry blotting. These conditions yield complete and extremely rapid transfer, with some systems completing transfer in 3–10 min. Such rapid blotting systems do not incorporate external cooling mechanisms, so the high power dissipation may generate more heat than other techniques. Rapid blotting systems are intended for extremely rapid transfers where heat-induced protein denaturation will not affect downstream applications.

Choose the right semi-dry or rapid transfer system based on the demands of your application.

 

Microfiltration (Dot Blotting)

Simple bulk transfer of proteins that are in solution may be achieved by manual application (dotting) to a membrane from a pipet or syringe, or by vacuum-assisted microfiltration. Manual dot-blotting with a pipet or syringe is generally used for small sample volumes. Microfiltration devices, on the other hand, enable application of larger volumes, multiple assays with different probes, and quick, reproducible screening of a large number of samples. Microfiltration facilitates the determination of working conditions for a new blotting assay and is a convenient method in any other situation where the resolving power of gel electrophoresis is not needed.

Comparison of electrophoretic protein transfer systems.

  Tank Blotting Semi-Dry Blotting
    Traditional Rapid
Transfer Time 30 min–overnight 15–60 min 3–10 min
Handling convenience Manual assembly of transfer components Manual assembly of transfer components Prepackaged, presaturated components
Transfer parameters Widest range of power settings and transfer times Power and transfer time limited due to lack of cooling options Preinstalled, customizable programs for transfer of most proteins, user-programmable settings for traditional semi-dry techniques
Molecular weight range Broad range Best for 30–120 kD Broad range
Temperature control Cooling with ice pack or refrigerated water recirculator None None
Buffer requirement 1–12 L, system-dependent 250 ml per blot No additional buffer required
 

References

Bjerrum OJ and Schafer-Nielsen C (1986). Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In Electrophoresis '86: Proceedings of the Fifth Meeting of the International Electrophoresis Society, M.J. Dunn, ed. (Weinheim, Germany: Wiley-VCH Verlag GmbH), pp. 315–327.

Burnette WN (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195–203.

Gershoni JM (1985). Protein blotting: developments and perspectives. Trends Biochem Sci 10, 103–106.

Kyhse-Andersen J (1984). Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10, 203–209.

Tovey ER and Baldo BA (1987). Comparison of semi-dry and conventional tank-buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes. Electrophoresis 8, 384–387.

Towbin H et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4354.

 

Related Content

 
Literature
Number Description Download
2895 Protein Blotting Guide, Ver C Click to download
2032 Western Blotting Detection Reagents Brochure, Rev F Click to download
 
 
LUSPPSESH [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [54.224.11.137, 10.232.3.215] [accept] = [text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8] [seourl] = [/en-us/applications-technologies/protein-blotting-methods] [x-amzn-trace-id] = [Root=1-5b757864-f8722fccbbbbd3746f7e5ad4] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=LUSPPSESH] [host] = [10.232.16.28:1776] [x-request-uri] = [/en-us/applications-technologies/protein-blotting-methods] [connection] = [Keep-Alive] [accept-encoding] = [gzip] [user-agent] = [CCBot/2.0 (https://commoncrawl.org/faq/)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/protein-blotting-methods LSR LUSPPSESH Protein Blotting Methods Protein Blotting Methods /webroot/web/html/lsr/solutions/technologies/western_blotting <p>The initial step in any blotting experiment is the transfer of proteins from a solution or gel and immobilization of those proteins on a solid membrane support. Immobilization of proteins on a membrane makes the proteins accessible to probes for specific proteins and enables quantitative detection. This section describes the principle of electrophoretic transfer of proteins, and details tank blotting, semi-dry blotting, and microfiltration or dot-blotting methods.</p> <p><strong>Related Topics</strong>: <a href="/evportal/destination/solutions?catID=LUSPQXC4S">Protein Blotting Equipment</a>, <a href="/evportal/destination/solutions?catID=LUSPSJ8UU">Membranes and Blotting Paper</a>, <a href="/evportal/destination/solutions?catID=LUSPTIMNI">Transfer Conditions</a>, <a href="/evportal/destination/solutions?catID=LUSPULKSY">Protein Blotting Detection and Imaging</a>, <a href="/evportal/destination/solutions?catID=LUSQA88UU">Transfer Buffers</a>, and <a href="/evportal/destination/solutions?catID=LUSQON470">Imaging and Analysis</a>.</p> Protein Blotting Methods <p>The two most common methods for protein transfer are:</p> <ul> <li><strong>Electrophoretic transfer</strong> &mdash; proteins separated in gels (for example, following polyacrylamide gel electrophoresis, or PAGE), are transferred onto membranes by electrophoretic transfer</li> <li><strong>Microfiltration (dot blotting)</strong> &mdash; proteins in solution are bound to membranes by vacuum driven microfiltration. This rapid approach is useful for determining working conditions for a new blotting assay or any other situation where the resolving power of gel electrophoresis is not needed</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Protein Transfer Methods <p><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/pet21_protein_transfer_methods.gif" alt="Protein Transfer Methods" /></p> <p class="caption"><strong>Protein transfer methods.</strong></p> <div class="top"><a href="#helptop">Back to Top</a></div> Electrophoretic Transfer <p><strong>Electrophoretic Transfer Setup</strong><br /> In electrophoretic transfer, an electric field is used to elute proteins from gels and transfer them to membranes. Electrophoretic transfer is the most widely used blotting method because of its speed and precision in replicating the pattern of separated proteins from a gel to a membrane.</p> <p>In an electrophoretic transfer, the membrane and protein-containing gel are placed together, with filter paper between two electrodes.</p> <p><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/pet21_gel_membrane_setup.gif" alt="Gel and membrane setup for electrophoretic transfer." width="385px" height="282px" /></p> <p class="caption"><strong>Gel and membrane setup for electrophoretic transfer.</strong></p> <p><strong>Electrophoretic Transfer Principle</strong></p> <p>Proteins migrate to the membrane following a current (I) that is generated by applying a voltage (V) across the electrodes* following Ohm's law:</p> <p style="margin-left:25px; font-weight: bold; font-size: larger;">V = I x R</p> <p>R is the resistance generated by the materials placed between the electrodes (that is, the transfer buffer, gel, membrane, and filter papers).</p> <p>The electric field strength (E, measured in V/cm) that is generated between the electrodes is the driving force for transfer. Both the applied voltage and the distance between the electrodes then play a major role in governing the rate of elution of the proteins form the gel. A number of other factors, including the size, shape, and charge of the protein*, the pH, viscosity, and ionic strength of the transfer buffer, and the composition of the gel, also influence the elution of particular proteins from gels.</p> <p>There are practical limits on field strength, however, due to the production of heat during transfer. The heat generated (Joule heating) is proportional to the power consumed by the electrical elements (P), which is equal to the product of the current (I) and voltage (V):</p> <p style="margin-left:25px; font-weight: bold; font-size: larger;">P = I x V = I<sup>2</sup> x R</p> <p>Joule heating increases temperature and decreases resistance of the transfer buffer. Such changes in resistance may lead to inconsistent field strength and transfer, or may cause the transfer buffer to lose its buffering capacity. In addition, excessive heat may cause the gel to deteriorate and stick to the membrane. The major limitation of any electrophoretic transfer method is the ability of the chamber to dissipate heat.</p> <p class="caption">* Proteins denatured with sodium dodecyl sulfate (SDS) carry a net negative charge and migrate toward the anode.</p> <p><strong>Electrophoretic Transfer Systems</strong><br /> There are two main types of electrophoretic blotting apparatus and transfer procedures (see <a href="#comparison">table below</a>):</p> <ul> <li><strong>Tank transfer systems</strong> &mdash; gels and membranes are submerged in transfer buffer in tanks; these systems are useful for most routine protein work, for efficient and quantitative protein transfers, and for transfers of proteins of all sizes. Tank transfer systems offer the most flexibility in choosing voltage settings, blotting times, and cooling options</li> <li><strong>Semi-dry systems</strong> &mdash; gels and membranes are sandwiched between buffer-saturated filter papers that are in direct contact with plate electrodes; these systems are typically easier to set up than tank systems and are useful when high-throughput is necessary and extended transfer times are not required, or when discontinuous buffer systems are used. Active cooling options are limited with semi-dry blotting</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Tank Blotting <p>In tank blotting systems, the gel and membrane sandwich is entirely submerged under transfer buffer within a buffer tank. A non-conducting cassette holds the membrane in close contact with the gel and the cassette assembly is placed in the tank between the electrodes, transverse to the electrical field, and submerged under conducting transfer buffer (Burnette 1981, Gershoni et al. 1985, Towbin et al. 1979). Although the large volumes of buffer in the tank dissipate the heat generated during transfer and provide the conducting capacity for extended transfer conditions, additional cooling mechanisms are offered by the various <a href="/evportal/destination/solutions?catID=LUSPQXC4S">tank blotting systems</a>.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Semi-Dry Blotting <p>In a semi-dry transfer, the gel and membrane are sandwiched between two stacks of filter paper that are in direct contact with plate electrodes (Bjerrum and Schafer-Nielsen 1986, Kyhse-Andersen 1984, Tovey and Baldo 1987). The term semi-dry refers to the limited amount of buffer, which is confined to the two stacks of filter paper.</p> <p>In semi-dry systems, the distance between the electrodes is limited only by the thickness of the gel and membrane sandwich. As a result, high electric field strengths and high-intensity blotting conditions are achieved. Under semi-dry conditions, some small proteins may be driven through the membrane in response to the high field strengths. Moreover, because low buffer capacity limits run times, some large proteins may be poorly transferred. Use of a discontinuous buffer system, that utilizes different buffer formulations on the anode and the cathode side of the transfer sandwich (see <a href="/evportal/destination/solutions?catID=LUSQA88UU">Transfer Buffers</a>) may enhance semi-dry transfer of high molecular weight proteins (&gt;80 kD). As semi-dry transfers require considerably less buffer and are easier to set up than the tank method, laboratories performing large numbers of blots often favor them.</p> <p>Novel buffer and material formulations have been developed that can be used with higher electric field strengths than those used in typical semi-dry blotting. These conditions yield complete and extremely rapid transfer, with some systems completing transfer in 3&ndash;10 min. Such rapid blotting systems do not incorporate external cooling mechanisms, so the high power dissipation may generate more heat than other techniques. Rapid blotting systems are intended for extremely rapid transfers where heat-induced protein denaturation will not affect downstream applications.</p> <p>Choose the right <a href="http://www.bio-rad.com/evportal/destination/product?catID=5290044b-3d9b-4a5e-987b-9c3214086bd4">semi-dry or rapid transfer system</a> based on the demands of your application.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Microfiltration (Dot Blotting) <p>Simple bulk transfer of proteins that are in solution may be achieved by manual application (dotting) to a membrane from a pipet or syringe, or by vacuum-assisted microfiltration. Manual dot-blotting with a pipet or syringe is generally used for small sample volumes. Microfiltration devices, on the other hand, enable application of larger volumes, multiple assays with different probes, and quick, reproducible screening of a large number of samples. Microfiltration facilitates the determination of working conditions for a new blotting assay and is a convenient method in any other situation where the resolving power of gel electrophoresis is not needed.</p> <p><a name="comparison"></a></p> <p><strong>Comparison of electrophoretic protein transfer systems.</strong></p> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td style="width: 149px;">&nbsp;</td> <td style="width: 149px;" align="center"><strong>Tank Blotting</strong></td> <td style="width: 298px;" colspan="2" align="center"><strong>Semi-Dry Blotting</strong></td> </tr> <tr> <td>&nbsp;</td> <td>&nbsp;</td> <td style="width: 149px;" align="center"><strong>Traditional</strong></td> <td style="width: 149px;" align="center"><strong>Rapid</strong></td> </tr> <tr> <td style="border-top:1px solid #000" valign="top"><strong>Transfer Time</strong></td> <td style="border-top:1px solid #000" valign="top">30 min&ndash;overnight</td> <td style="border-top:1px solid #000" valign="top">15&ndash;60 min</td> <td style="border-top:1px solid #000" valign="top">3&ndash;10 min</td> </tr> <tr class="pd_colorbackground"> <td valign="top"><strong>Handling convenience</strong></td> <td valign="top">Manual assembly of transfer components</td> <td valign="top">Manual assembly of transfer components</td> <td valign="top">Prepackaged, presaturated components</td> </tr> <tr> <td valign="top"><strong>Transfer parameters</strong></td> <td valign="top">Widest range of power settings and transfer times</td> <td valign="top">Power and transfer time limited due to lack of cooling options</td> <td valign="top">Preinstalled, customizable programs for transfer of most proteins, user-programmable settings for traditional semi-dry techniques</td> </tr> <tr class="pd_colorbackground"> <td valign="top"><strong>Molecular weight range</strong></td> <td valign="top">Broad range</td> <td valign="top">Best for 30&ndash;120 kD</td> <td valign="top">Broad range</td> </tr> <tr> <td valign="top"><strong>Temperature control</strong></td> <td valign="top">Cooling with ice pack or refrigerated water recirculator</td> <td valign="top">None</td> <td valign="top">None</td> </tr> <tr class="pd_colorbackground"> <td style="border-bottomx:1px solid #000" valign="top"><strong>Buffer requirement</strong></td> <td style="border-bottomx:1px solid #000" valign="top">1&ndash;12 L, system-dependent</td> <td style="border-bottomx:1px solid #000" valign="top">250 ml per blot</td> <td style="border-bottomx:1px solid #000" valign="top">No additional buffer required</td> </tr> </tbody> </table> <div class="top"><a href="#helptop">Back to Top</a></div> References <p>Bjerrum OJ and Schafer-Nielsen C (1986). Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In Electrophoresis '86: Proceedings of the Fifth Meeting of the International Electrophoresis Society, M.J. Dunn, ed. (Weinheim, Germany: Wiley-VCH Verlag GmbH), pp. 315&ndash;327.</p> <p>Burnette WN (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195&ndash;203.</p> <p>Gershoni JM (1985). Protein blotting: developments and perspectives. Trends Biochem Sci 10, 103&ndash;106.</p> <p>Kyhse-Andersen J (1984). Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10, 203&ndash;209.</p> <p>Tovey ER and Baldo BA (1987). Comparison of semi-dry and conventional tank-buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes. Electrophoresis 8, 384&ndash;387.</p> <p>Towbin H et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350&ndash;4354.</p> <div class="top"><a href="#helptop">Back to Top</a></div> 2895 2032 Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Mini Format 1D-Electrophoresis Systems/Mini-PROTEAN Precast Gels/Mini-PROTEAN TGX Stain-Free Precast Gels ->MTS::L0U6RK15##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Semi-Dry Blotting Systems/Trans-Blot Turbo Transfer System ->MTS::LGOQBW15##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Western Blotting Membranes and Filter Paper/Immun-Blot PVDF (for Western Blotting) ->MT::9f95f7dc-8ad2-4923-8eb4-be822b64e2f4##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Immunodetection Reagents and Kits/Immun-Star HRP Chemiluminescence Kits ->MT::e08909eb-3369-42ec-a585-df189004268f## Life Science Research/Solutions/Technologies/Protein Electrophoresis ->MTS::LUSOVO47B##Life Science Research/Solutions/Technologies/Protein Electrophoresis/Protein Detection and Analysis ->MTS::LUSPLU15## Life Science Research/Solutions/Applications/Protein Separation and Analysis/Fast Separation Transfer and Analysis ->MTS::LUSOUMFCN## Eddie C Protein Blotting Methods 12/08/11 10:59 AM 12/08/21 11:02 AM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/Western_Blotting N 0
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