Western Blot Doctor™ — Protein Band Size and Pattern Problems

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Overview

The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. In this section, you can find solutions to issues related to protein band size and pattern problems.

Other sections in the Western Blot Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem.

Band(s) at significantly higher MW than expected

A blot with non-specific-bands – Western Blot Doctor

Multiple bands at various MWs

 

Problem: Band(s) at lower MW than expected

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Possible sources of unexpectedly low-MW bands include protein cleavage or degradation, splice variants, and nontarget proteins bearing similar epitopes.
Possible causes: Solutions:
Target protein has been cleaved or digested
  • Use a fresh sample that has been kept on ice
  • Add fresh protease inhibitors to the lysis buffer (e.g., EDTA or PMSF)
Splicing variant exists
  • Confirm whether a splice variant may exist for your protein
  • Try an alternate antibody
 Bio-Rad now offers high-quality antibodies for all applications
Another protein bearing the same/similar epitope is detected by the antibody
  • Make sure you include a negative control for the expression of your protein
  • Try an alternate antibody
Bio-Rad now offers PrecisionAb™ Validated Western Blotting Antibodies for superior performance in western blot detection
 

Problem: Band(s) at slightly higher MW than expected or blurry

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Bands at MW slightly higher than expected and/or blurred may indicate protein modifications such as glycosylation.
Possible causes: Solutions:
Protein may be glycosylated or otherwise modified at one or more amino acid residues
  • Use enzymes that remove suspected modification to restore molecular weight closer to expected
  • Check amino acid sequence for known motifs for posttranslational modifications, and search literature for other evidence of modified forms
 

Problem: Band(s) at significantly higher MW than expected

Back to Top
Sources of unexpectedly high-MW bands include protein-protein interactions and antibody cross-reactivity.
Possible causes: Solutions:
Dimers, multimers, or protein-protein interactions occurring because samples have not been fully reduced or denatured.
  • Add fresh DTT or 2-mercaptoethanol to samples and reheat before repeating experiment to remove disulfide bonds
  • Try stronger reducing agents e.g., tryibutylpohsphine or TCEP
  • Add a chaotropic denaturant (e.g., urea) to denature the protein
  • Prepare new samples with fresh loading buffer
 

Problem: Multiple bands at various MWs

Back to Top
Multiple nonspecific bands on the blot may be due to antibodies of poor quality or at too high a concentration, insufficient blocking, or nonspecific binding due to the presence of SDS. A blot with non-specific-bands – Western Blot Doctor
Possible causes: Solutions:
Primary antibody concentration too high or cross-reactivity with similar epitopes on other proteins
Secondary antibody concentration too high, leading to nonspecific binding
  • Decrease or optimize the concentration of the secondary antibody, e.g., using a checkerboard screening protocol
  • Use an affinity-purified secondary antibody
  • Repeat immunodetection with secondary antibody alone to check for nonspecific binding
Protein exists in several different isoforms
  • Check research literature for existence of isoforms or variants
Primary or secondary antibody contaminated with nonspecific IgG or with IgG cross-reactive among species

 

  • Use purified IgG primary antibody fractions and affinity-purified blotting-grade cross-adsorbed secondary antibody
Monoclonal antibodies reacted nonspecifically with SDS-denatured proteins
  • Compare the binding of other monoclonal or polyclonal antibodies
  • Blot native proteins as a comparison, e.g., by blue native PAGE
Nonspecific interactions occurring due to ionic associations; for example, avidin, a glycosylated protein, may bind to more acidic proteins on blots
  • Increase washing stringency:
    • Increase the ionic strength of the incubation buffers
      1. Increase the salt concentration of your TBS-T
      2. Try PBS-T instead of TBS-T (do not do this if using phosphospecific antibodies)
    • Increase the duration of washes
    • Increase the number of washes
    • Perform washes at room temperature
  • Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween-20
  • Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding
  • Increase the Tween-20 concentration to 0.01–0.5% (v/v)
Insufficient blocking of nonspecific sites
  • Increase the concentration of blocking reagent (e.g., BSA, nonfat dry milk, etc.) from 5% to 7% (w/v)
  • Consider blocking overnight at 4ºC or at least 1 hour at room temp (increase length of incubations if necessary)
  • If not already included, add up to 0.01–0.5% Tween-20 to blocking buffer
  • Prepare antibody dilutions to the same blocking buffer with same increased concentration of Tween-20
SDS caused nonspecific antibody binding to immobilized proteins
  • Be sure to equilibrate gel with transfer buffer before transfer
  • If the transfer buffer contains SDS, be sure to include a wash step before performing the first antibody incubation step; wash step can be performed with washing buffer
  • If washing does not resolve the problem, consider avoiding SDS during blotting procedure if possible
 

 
 
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    MIW4MCKG4 [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [54.196.213.0, 10.232.19.41] [accept] = [text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8] [seourl] = [/en-us/applications-technologies/western-blot-doctor-protein-band-size-pattern-problems] [x-amzn-trace-id] = [Root=1-5a910baf-76eeb1621676ce6348f2b561] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=MIW4MCKG4] [host] = [10.232.16.28:1776] [x-request-uri] = [/en-us/applications-technologies/western-blot-doctor-protein-band-size-pattern-problems] [connection] = [Keep-Alive] [accept-encoding] = [x-gzip, gzip, deflate] [user-agent] = [CCBot/2.0 (http://commoncrawl.org/faq/)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/western-blot-doctor-protein-band-size-pattern-problems MIW4MCKG4 Protein Transfer Western Blot Doctor&#153; — Protein Band Size and Pattern Problems /webroot/web/html/lsr/solutions/technologies/western_blotting <p>The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. In this section, you can find solutions to issues related to protein band size and pattern problems.</p> <p><strong>Other sections in the Western Blot Doctor:</strong></p> <ul> <li><a href="/en-us/applications-technologies/western-blot-doctor-protein-band-appearance-problems">Band Appearance Problems</a></li> <li><a href="/en-us/applications-technologies/western-blot-doctor-blot-background-problems">Blot Background Problems</a></li> <li><a href="/en-us/applications-technologies/western-blot-doctor-signal-strength-problems">Signal Strength Problems</a></li> <li>Band Size and Pattern Problems</li> </ul> <div class="contentroundwrap"> <div class="contentroundtop"> <h3>Problems and Solutions</h3> </div> <div class="contentroundmidwrap"> <div class="contentroundmid gels"> <blockquote> <p>Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem.</p> </blockquote> <table class="pd_table" border="0"> <tbody> <tr> <td valign="top"> <div><a href="#lowermw"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/Bands-at-lower-MW-than-expected.jpg" alt="" width="120" height="120" /></a> <p class="caption"><a href="#lowermw">Band(s) at lower MW than expected</a></p> </div> </td> <td valign="top"> <div><a href="#slightlyhighermw"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/Bands at slightly higher MW than expected.jpg" alt="" width="120" height="120" /></a> <p class="caption"><a href="#slightlyhighermw">Band(s) at slightly higher MW than expected, and may be blurred</a></p> </div> </td> <td valign="top"><a href="#significantlyhighermw"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/Bands-at-significantly-higher-mw-than-expected.jpg" alt="" width="120" height="120" /></a> <p class="caption"><a href="#significantlyhighermw">Band(s) at significantly higher MW than expected</a></p> </td> <td valign="top"><a href="#uneventransfer"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/non-specific-bands-thumb-western-blot-doctor-non-specific-bands.jpg" alt="A blot with non-specific-bands &ndash; Western Blot Doctor" width="120" height="120" /></a> <p class="caption"><a href="#significantlyhighermw">Multiple bands at various MWs</a></p> </td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <div class="contentroundwrap"><a name="lowermw"></a> <div class="contentroundtop"> <h3>Problem: Band(s) at lower MW than expected</h3> <a href="#helptop">Back to Top</a></div> </div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td valign="top">Possible sources of unexpectedly low-MW bands include protein cleavage or degradation, splice variants, and nontarget proteins bearing similar epitopes.</td> <td valign="top"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/Bands-at-lower-MW-than-expected.jpg" alt="" width="270" height="215" /></td> </tr> <tr class="pd_colorbackground"> <td width="28%" valign="top"><strong>Possible causes:</strong></td> <td width="70%"><strong>Solutions:</strong></td> </tr> <tr> <td valign="top">Target protein has been cleaved or digested</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Use a fresh sample that has been kept on ice</li> <li style="margin-left:-30px; font-size:11px">Add fresh protease inhibitors to the lysis buffer (e.g., EDTA or PMSF)</li> </ul> </td> </tr> <tr class="pd_colorbackground"> <td valign="top">Splicing variant exists</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px;font-size:11px">Confirm whether a splice variant may exist for your protein <strong></strong></li> <li style="margin-left:-30px; font-size:11px">Try an alternate antibody</li> </ul> &nbsp;<a href="https://www.abdserotec.com">Bio-Rad now offers high-quality antibodies</a> for all applications<br /></td> </tr> <tr> <td valign="top">Another protein bearing the same/similar epitope is detected by the antibody</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Make sure you include a negative control for the expression of your protein</li> <li style="margin-left:-30px; font-size:11px">Try an alternate antibody</li> </ul> Bio-Rad now offers <a href="/en-us/product/precisionab-validated-western-blotting-antibodies">PrecisionAb&trade; Validated Western Blotting Antibodies</a> for superior performance in western blot detection</td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> <br /> <div class="contentroundwrap"><a name="slightlyhighermw"></a> <div class="contentroundtop"> <h3>Problem: Band(s) at slightly higher MW than expected or blurry<br /></h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td valign="top">Bands at MW slightly higher than expected and/or blurred may indicate protein modifications such as glycosylation.</td> <td valign="top"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/Bands at slightly higher MW than expected.jpg" alt="" width="270" height="215" /></td> </tr> <tr class="pd_colorbackground"> <td width="28%" valign="top"><strong>Possible causes:</strong></td> <td width="70%"><strong>Solutions:</strong></td> </tr> <tr> <td width="28%" valign="top">Protein may be glycosylated or otherwise modified at one or more amino acid residues</td> <td width="70%" valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Use enzymes that remove suspected modification to restore molecular weight closer to expected</li> <li style="margin-left:-30px; font-size:11px">Check amino acid sequence for known motifs for posttranslational modifications, and search literature for other evidence of modified forms</li> </ul> </td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <br /> <div class="contentroundwrap"><a name="significantlyhighermw"></a> <div class="contentroundtop"> <h3>Problem: Band(s) at significantly higher MW than expected</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td valign="top">Sources of unexpectedly high-MW bands include protein-protein interactions and antibody cross-reactivity.</td> <td valign="top"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/Bands-at-significantly-higher-mw-than-expected.jpg" alt="" width="270" height="215" /></td> </tr> <tr class="pd_colorbackground"> <td width="28%" valign="top"><strong>Possible causes:</strong></td> <td width="70%" valign="top"><strong>Solutions:</strong></td> </tr> <tr> <td width="28%" valign="top">Dimers, multimers, or protein-protein interactions occurring because samples have not been fully reduced or denatured.</td> <td width="70%" valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Add fresh DTT or 2-mercaptoethanol to samples and reheat before repeating experiment to remove disulfide bonds </li> <li style="margin-left:-30px; font-size:11px">Try stronger reducing agents e.g., tryibutylpohsphine or TCEP</li> <li style="margin-left:-30px; font-size:11px">Add a chaotropic denaturant (e.g., <a href="/en-us/product/running-buffers-reagents?tab=Ordering">urea</a>) to denature the protein</li> <li style="margin-left:-30px; font-size:11px">Prepare new samples with fresh loading buffer</li> </ul> </td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <br /> <div class="contentroundwrap"><a name="uneventransfer"></a> <div class="contentroundtop"> <h3>Problem: Multiple bands at various MWs</h3> <a href="#helptop">Back to Top</a></div> <div class="contentroundmidwrap"> <div class="contentroundmid"> <table class="pd_table pd_gridlines" border="0"> <tbody> <tr> <td valign="top">Multiple nonspecific bands on the blot may be due to antibodies of poor quality or at too high a concentration, insufficient blocking, or nonspecific binding due to the presence of SDS.</td> <td valign="top"><img style="float: left;" src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/non-specific-bands-western-blot-doctor-non-specific-bands.jpg" alt="A blot with non-specific-bands &ndash; Western Blot Doctor" width="270" height="215" /></td> </tr> <tr class="pd_colorbackground"> <td width="28%" valign="top"><strong>Possible causes:</strong></td> <td width="70%"><strong>Solutions:</strong></td> </tr> <tr> <td valign="top">Primary antibody concentration too high or cross-reactivity with similar epitopes on other proteins</td> <td width="70%" valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Use an affinity-purified primary antibody</li> <li style="margin-left:-30px; font-size:11px">Optimize primary antibody concentration</li> <li style="margin-left:-30px; font-size:11px">Try an alternate antibody. Bio-Rad now offers <a href="/en-us/product/precisionab-validated-western-blotting-antibodies">PrecisionAb&trade; Validated Western Blotting Antibodies</a> for superior performance in western blot detection <ul> <li style="margin-left:-30px; font-size:11px">Check antibody specificity with a blocking peptide (pre-incubate the antibody with an excess of the same sequence used to generate the antibody; see <a href="https://www.abdserotec.com/western-blot-demonstrating-antibody-specificity.html">Demonstrating Antibody Specificity</a>)</li> </ul> </li> </ul> </td> </tr> <tr class="pd_colorbackground"> <td valign="top">Secondary antibody concentration too high, leading to nonspecific binding</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Decrease or optimize the concentration of the secondary antibody, e.g., using a checkerboard screening protocol </li> <li style="margin-left:-30px; font-size:11px">Use an affinity-purified secondary antibody</li> <li style="margin-left:-30px; font-size:11px">Repeat immunodetection with secondary antibody alone to check for nonspecific binding</li> </ul> </td> </tr> <tr> <td valign="top">Protein exists in several different isoforms</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Check research literature for existence of isoforms or variants</li> </ul> </td> </tr> <tr class="pd_colorbackground"> <td valign="top">Primary or secondary antibody contaminated with nonspecific IgG or with IgG cross-reactive among species <p>&nbsp;</p> </td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Use purified IgG primary antibody fractions and affinity-purified blotting-grade cross-adsorbed secondary antibody</li> </ul> </td> </tr> <tr> <td valign="top">Monoclonal antibodies reacted nonspecifically with SDS-denatured proteins</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Compare the binding of other monoclonal or polyclonal antibodies</li> <li style="margin-left:-30px; font-size:11px">Blot native proteins as a comparison, e.g., by <a href="/en-us/applications-technologies/protein-electrophoresis-methods#4">blue native PAGE</a></li> </ul> </td> </tr> <tr class="pd_colorbackground"> <td valign="top">Nonspecific interactions occurring due to ionic associations; for example, avidin, a glycosylated protein, may bind to more acidic proteins on blots</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Increase washing stringency: <ul> <li style="margin-left:-30px; font-size:11px">Increase the ionic strength of the incubation buffers<ol> <li style="margin-left:-30px; font-size:11px">Increase the salt concentration of your TBS-T</li> <li style="margin-left:-30px; font-size:11px">Try PBS-T instead of TBS-T (do not do this if using phosphospecific antibodies)</li> </ol></li> <li style="margin-left:-30px; font-size:11px">Increase the duration of washes</li> <li style="margin-left:-30px; font-size:11px">Increase the number of washes</li> <li style="margin-left:-30px; font-size:11px">Perform washes at room temperature</li> </ul> </li> <li style="margin-left:-30px; font-size:11px">Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween-20</li> <li style="margin-left:-30px; font-size:11px">Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding</li> <li style="margin-left:-30px; font-size:11px">Increase the Tween-20 concentration to 0.01&ndash;0.5% (v/v)</li> </ul> </td> </tr> <tr> <td valign="top">Insufficient blocking of nonspecific sites</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Increase the concentration of blocking reagent (e.g., BSA, nonfat dry milk, etc.) from 5% to 7% (w/v)</li> <li style="margin-left:-30px; font-size:11px">Consider blocking overnight at 4&ordm;C or at least 1 hour at room temp (increase length of incubations if necessary)</li> <li style="margin-left:-30px; font-size:11px">If not already included, add up to 0.01&ndash;0.5% Tween-20 to blocking buffer</li> <li style="margin-left:-30px; font-size:11px">Prepare antibody dilutions to the same blocking buffer with same increased concentration of Tween-20</li> </ul> </td> </tr> <tr class="pd_colorbackground"> <td valign="top">SDS caused nonspecific antibody binding to immobilized proteins</td> <td valign="top"> <ul> <li style="margin-left:-30px; margin-top:-10px; font-size:11px">Be sure to equilibrate gel with transfer buffer before transfer</li> <li style="margin-left:-30px; font-size:11px">If the transfer buffer contains SDS, be sure to include a wash step before performing the first antibody incubation step; wash step can be performed with washing buffer</li> <li style="margin-left:-30px; font-size:11px">If washing does not resolve the problem, consider avoiding SDS during blotting procedure if possible</li> </ul> </td> </tr> </tbody> </table> </div> </div> <div class="contentroundbot">&nbsp;</div> </div> <br /> Karen Moss Western Blot Doctor - Band Size and Pattern Problems western, blot, blotting, doctor, troubleshoot, troubleshooting, self-help, guide, band, bands, nonspecific, non-specific, non, specific, size, sizes, pattern, patterns, problem, problems, lower, mw, molecular, weight, slightly, higher, blurred, blurr, significantly, significant, multiple, dtt, 2-mercaptoethanol, 2, mercaptoethanol, reducing, agent, urea, pbs-t, tbs-t, tbs-tween, tbs, tween, tween-20, sds, over-night, overnight, over, night, blocking, buffer, polyclonal, monoclonal, mouse, rabbit, primary, secondary, antibody, antibodies, ddt, tryibutylpohsphine, tcep, edta 02/28/13 09:28 AM 02/28/23 09:29 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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