Western Blotting Transfer Techniques

 

Better Transfer

Efficient transfer of proteins from gel to membrane.

Sign Up for Western Blotting Tips and Webinar Alerts

 

Transfer Basics

Protein Transfer on to a Membrane

In western blotting, following electrophoresis is the transfer step. This step consists of moving the proteins from a gel matrix to a synthetic membrane support where it is bound, forming the blot.



 

Protein Blotting Workflow

  • Protein Blotting Workflow: method and equipment selection for protein transfer

    Select Method and Equipment for Transfer

    Select from either traditional tank transfer, semi-dry, or advanced rapid semi-dry transfer methods.

  • Protein Blotting Workflow: reagents and transfer buffer preparation

    Prepare Transfer Buffer and Reagents

    Prepare transfer buffer sufficient for the transfer cell and for equilibration of gels and membranes.

    Note that rapid semi-dry systems use specialized buffers and may not need gel equilibration.

  • Protein Blotting Workflow: gel and membrane equilibration

    Equilibrate Gel and Membrane

    Equilibrate gels and membranes in transfer buffer if necessary.

  • Protein Blotting Workflow: gel and membrane sandwich assembly

    Assemble Gel and Membrane Sandwich

    Place gel and membrane between buffer-soaked filter papers.

  • Protein Blotting Workflow: protein transfer

    Perform Transfer

    Place transfer sandwich in transfer cell and fill with buffer. Select transfer conditions and begin transfer.

Electrophoretic Transfer

Gel and membrane set up for electrophoretic transfer — gel membrane sandwich.

Gel and Membrane Setup for
Electrophoretic Transfer

The most common method of transfer in western blotting is electrophoretic transfer, where an electric field is used to elute proteins from gels and transfer them to membranes. During this process, the membrane and gel are placed together, with filter paper between two electrodes. Voltage is applied between the electrodes and proteins migrate to the membrane following the current that is generated by the applied voltage across the electrodes.

The electric field strength (measured in V/cm) that is generated between the electrodes is the driving force for transfer. Both the voltage and the distance between the electrodes then play a major role in governing the rate of elution of the proteins from the gel. Transfer conditions must be optimized for proteins of different molecular weights, e.g., to prevent under-transfer (incomplete transfer of proteins out of the gel) or over-transfer (loss of proteins passing completely through the membrane).

Transfer Methods

There are three main types of transfer apparatus and procedures:
tank transfer systems, semi-dry systems, and rapid-transfer systems.

  • Tank Transfer Systems

    These systems are useful for most routine protein work and for transfers of proteins of all sizes; gels and membranes are submerged under transfer buffer in tanks.

    Diagram of a tank transfer system
  • Diagram of a semi-dry system

    Semi-Dry Systems

    Gels and membranes are sandwiched between buffer-wetted filter papers that are in direct contact with flat-plate electrodes. These systems are typically easier to set up than tank systems.

  • Rapid-Transfer Systems

    A recent development, these systems utilize specialized apparatus that use proprietary filter papers and specialized buffers to rapidly and efficiently transfer proteins. Filter papers and membranes are pre-wet and packaged in single-use packages, simplifying assembly of the transfer stack.

    Diagram of a rapid-transfer system

Comparison of Western Blotting 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 transfers 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

See Our Western Blotting Transfer Systems »

Transfer Buffers

Transfer buffers contain a conductive, strong buffering agent (for example, Tris, CAPS, or carbonate) in order to maintain the conductivity and pH of the system during transfer. In addition, alcohol (methanol or ethanol) may be included in the transfer buffer to promote binding of proteins to membranes, and SDS may be added to promote elution of proteins from gels.

Compatible Transfer Buffer by Gel Type

Gel Type Transfer Buffer Western Blotting Transfer System
SDS-PAGE Towbin with or without SDS, CAPS, carbonate, Bjerrum Schafer-Nielsen Tank blotting or semi-dry blotting
Tris-Tricine Towbin, CAPS Tank blotting recommended; needs high-capacity, small pore-size membrane; pH of buffer may be critical
Native, non-denaturing Depends on pH of gel buffer and pI of protein of interest Tank blotting recommended; temperature regulation may be needed to maintain activity
Acid urea 0.7% acetic acid Use acid-gel transfer protocol (membrane toward cathode)

SDS and Alcohol

SDS and alcohol play opposing roles in a transfer. SDS promotes elution of the protein from the gel and in cases where certain proteins are difficult to elute from the gel, SDS may be added to the transfer buffer. However, SDS in the transfer buffer decreases the binding efficiency of protein to nitrocellulose membrane; PVDF membrane can be substituted if desired.

Alcohol, on the other hand, removes the SDS from SDS-protein complexes and improves the binding of protein to nitrocellulose membrane. But alcohol may cause a reduction in the gel pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral.

 

Recommended Transfer Buffer by Application

Application Recommended Transfer Buffer
High-molecular-weight proteins Towbin with SDS
Small proteins and peptides Towbin, CAPS
Basic proteins (pI > 9) in denaturing gels CAPS, carbonate, Bjerrum Schafer-Nielsen
Basic proteins (pI > 9) in native or non-denaturing gels 0.7% acetic acid
Glycoproteins Towbin with or without SDS, CAPS, carbonate, Bjerrum Schafer-Nielsen nondenaturing gels
Proteoglycans Towbin, Bjerrum Schafer-Nielsen

See Our Western Blotting Buffers and Reagents »

  • Transfer Buffer Tips

    • Do not use the same batch of transfer buffer more than once, as the buffer will likely lose its capacity to maintain a stable pH during transfer
    • Do not dilute transfer buffers; this will also decrease buffering capacity
    • Do not adjust the pH of transfer buffers when not indicated, as this increases buffer conductivity, which is manifested by higher initial current output and decreased resistance

Membrane Types

Nitrocellulose, supported nitrocellulose, and PVDF membranes for protein transfer during western blotting

Nitrocellulose and Supported Nitrocellulose

Nitrocellulose membranes are easily wetted in water or transfer buffer, and it is compatible with a wide range of protein detection systems. Unsupported nitrocellulose is innately fragile and are not recommended for stripping and reprobing.

Supported nitrocellulose is an inert support structure with nitrocellulose applied to it. This extra support gives the membrane increased strength and resilience to withstand reprobing and autoclaving (121℃) while maintaining the ease of wetting of nitrocellulose.

Polyvinylidene Difluoride (PVDF)

PVDF membrane has a strong protein binding capacity (about twice that of nitrocellulose) and will not crack nor tear in common handling, so it can withstand repeated stripping and reprobing.

Low-fluorescence formulations of PVDF are available and combine the advantages of PVDF with low autofluorescence across a wide range of excitation and emission wavelengths. This low autofluorescence allows longer exposure times without increasing background fluorescence levels, allowing detection of faint signals.

Pore Size

Membranes are commonly available in two pore sizes:

  • 0.45 μm pore size membranes are recommended for most analytical blotting experiments
  • 0.2 μm pore size membranes are most suitable for transfer of low-molecular-weight (<15,000 kD) proteins that might move through larger membrane pores

See Our Western Blotting Transfer Membranes »

Power Conditions and Transfer Time

Factors Affecting Transfer Conditions

  • Buffer composition affecting protein transfer conditions

    Buffer composition

    Addition of SDS or changes in ion concentration due to addition of acid or base, changes the resistance of the transfer system and therefore current and results in changes in current and voltage readings as well as transfer efficiency.

  • Number of gels increases current, affecting protein transfer conditions

    Number of gels

    Current increases slightly as the number of gels increases. Consider additional cooling or limiting transfer time.

  • Higher gel acrylamide percentages slow down migration,  affecting protein transfer conditions

    Gel acrylamide percentage

    Higher acrylamide percentages slow protein migration out of the gel. Consider extending transfer time.

    Proteins move quickly through gels with low acrylamide percentages. Limit transfer time to prevent small proteins from "blowing through" membrane.

  • Increased buffer volume increases current, affecting protein transfer conditions

    Volume of buffer

    Current increases when volume increases. Consider additional cooling. Consider additional cooling or limiting transfer time.

  • Increased transfer temperature increases current, affecting protein transfer conditions

    Transfer temperature

    Current increases when temperature increases.

  • Large proteins migrate slowly, affecting protein transfer conditions

    Protein molecular weight

    Large proteins migrate more slowly out of the gel. Consider extending transfer time or increasing voltage.

    Conversely, small proteins migrate quickly and may be pushed through the membrane. Consider limiting transfer time or voltage.

For best transfer results, use the highest electric field strength possible within the heat dissipation capabilities of the system. During transfer, the buffer warms as a result of the power dissipated through the system, and its resistance drops. Such heating and changes to resistance may lead to inconsistent field strength and transfer, and may cause the transfer buffer to lose its buffering capacity, or may cause the gel to melt and stick to the membrane.

In general, transfer from thicker or higher percentage gels, or transfer of high-molecular-weight proteins require high electric fields or longer transfer times. Complete protein elution from the gel can be confirmed by post-staining the gel with a total protein stain. Conversely, transfer from thin, low-percentage gels or of proteins of low molecular weight require lower field strengths and shorter transfer times. In some cases, small proteins may “blow through” a membrane. This over-transfer can be detected by placing a second membrane behind the first and staining this membrane with a total protein stain or by imaging the backup membrane using a stain-free imaging system.

Power Settings and Transfer Time by Gel Type

SDS-PAGE Gels (Towbin Buffer)
  Standard (Overnight) High-Intensity
Trans-Blot Cell
plate electrodes
10 V/100 mA, 16 hr 50–100 V/700–1,600 mA, 30–60 min
Trans-Blot Cell
wire electrodes
30 V/100 mA, 16 hr 100–200 V/300–800 mA, 30 min–4 hr
Trans-Blot Plus Cell 30 V/0.5 A, 16 hr 100 V/1,500 mA, 60 min
Mini Trans-Blot Cell 30 V/90 mA, 16 hr 100 V/350 mA, 60 min
Criterion Blotter
plate electrodes
10 V/30–40 mA, 16 hr 100 V/750–1,000 mA, 30 min
Criterion Blotter
wire electrodes
10 V/50–80 mA, 16 hr 100 V/380–500 mA, 60 min
Trans-Blot SD Cell N/A Mini gels: 10–15 V/5.5 mA/cm2, 10–30 min
Large gels: 15–25 V/3 mA/cm2, 30–60 min
Trans-Blot Turbo System N/A Mini gels: 25 V/1,300 mA, 7 min
Midi gels: 25 V/2,500 mA, 7 min

Constant Voltage Transfers

If the voltage is held constant throughout a transfer, field strength remains constant, providing the most efficient transfer possible for tank blotting methods. However, the current in tank transfer systems increases as the resistance drops due to heating; in most semi-dry systems, current drops as a result of buffer depletion. Therefore, the overall power increases during transfer, and there is an increased risk of heating. Use of the cooling elements available with the various tank blotting systems helps prevent problems with heating.

Constant Current Transfers

If the current is held constant during a run, a decrease in resistance results in a decrease in voltage and power over time and therefore lower risk of heating. However, proteins are transferred more slowly due to decreased field strength.

See Our Western Blotting Power Supplies »

Tips for Better Transfers

  • Remove Bubbles

    Remove Bubbles.

    Uniform protein transfer requires complete contact between the components of the transfer stack, especially between the gel and membrane. Air bubbles cause blank spots on the membrane and proteins to “flow” around the air bubble, so use a gel roller and roll those air bubbles out! Tip: Gradient gels are best rolled up-down, rather than side-to-side. Side-to-side rolling causes gradient gels to warp and wrinkle.

  • Equilibrate before Transfer

    Equilibrate before Transfer.

    When performing traditional wet tank transfers and traditional semi-dry transfers, optimal results are obtained when the electrophoresis buffer saturating the gel is replaced with transfer buffer. Contaminating buffer salts increase the conductivity of the transfer buffer resulting in excess heat during the transfer. Rinse gels briefly in water then equilibrate the gel for 15 minutes and the membrane for at least 5 minutes in transfer buffer. Note that gel equilibration is not recommended for some rapid transfer systems, follow the manufacturer’s directions.

  • Do Not Allow Membranes to Dry Out

    Don't Allow Membranes to Dry Out.

    Transfer requires that the membrane stays wet in transfer buffer both before and after transfer. Heat generated during a transfer can dry out membranes once removed from the transfer stack. This is especially important with PVDF membranes since they are hydrophobic. Work quickly, and have a dish of buffer ready to immerse your membrane into in order to avoid drying the membrane. If they do dry, nitrocellulose can simply be re-wet in buffer while PVDF membranes will require re-activation in alcohol.

Western Blot Transfer Protocols and Resources

  • Protein Blotting Guide

    Protein Blotting Guide
    (PDF 7.9 MB)

    Details on blotting technology, methods, products, tips, techniques, and troubleshooting guidelines.

  • Western blot doctor — a resource to help troubleshoot western blotting issues.

    Western Blot Doctor

    Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look their best.

  • Transfer of High-Molecular–Weight Proteins

    Transfer of High-Molecular–Weight Proteins

    Learn more about the relative transfer efficiencies of tank, semi-dry, and rapid blotting systems for western blotting with large proteins.

  • Membrane Selection Guide

    Membrane Selection Guide

    Choose the right blotting membrane material, porosity, and form factor for your western blotting experiments.

Better Western Blotting Guide

Tips, Techniques, and Technologies from the Western Blotting Experts at Bio-Rad Laboratories

Get Your Free Guide

Western Blot University

More Western Blotting

Western Blotting University

Courses designed to make you a western blotting expert.

Monday – Friday, April 19 — April 23, 2021 at 9:00AM PDT (5:00PM BST)

Join us for an in-depth experience learning about western blotting principles and techniques. This five-part webinar series is for anyone who has ever asked how to perform a western blot and would like to have a detailed understanding of western blotting principles at each step of the workflow.

Register for Webinar Series