Guide to gels and transfer methods for the detection of high molecular weight proteins

Reliable transfer of high molecular weight (HMW) proteins (i.e., >150 kDa) from a gel to membrane during western blotting is a common challenge. Many factors affect the efficiency at which HMW proteins transfer. To identify key factors affecting the transfer efficiency of proteins from gel to membrane, we conducted a series of experiments to evaluate best practices when performing this workflow step in wet, rapid semi-dry, or rapid dry transfers.

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With respect to transfer efficiency, most modern transfer systems will transfer a broad range of protein molecular weights with high efficiency. Most modern semi-dry systems and dry systems offer preprogrammed optimized methods for particular molecular weight ranges, including high molecular weights. Transferring very high molecular weight proteins often requires optimization regardless of the system used.

Using the right gel for high molecular weight protein separation

Recommendation 1: Use Tris-acetate gels

Choosing the right gel is a key factor in the successful transfer of HMW proteins. When targeting HMW proteins for your transfer, it is best to use a Tris-acetate gel or low percentage Bis-Tris or Tris-glycine gel. While 4–20% Tris-glycine gradient gels are very popular because of their ability to separate a broad range of proteins (20–200 kDa), they are not recommended for separation of HMW proteins. Proteins >200 kDa are compacted into a very narrow region at the top of the running portion of the gel, leading to poor resolution of protein bands (Figure 1). By using a Tris-acetate or low gradient Bis-Tris or Tris-glycine gel, HMW proteins can migrate further through the gel, allowing increased distance between protein bands. The open matrix structure that allows the HMW proteins to migrate farther through the gel allows better transfer of the HMW proteins out of the gel leading to increased transfer efficiencies and higher sensitivity. A comparison of HMW protein separation using different gel chemistries and gradients shows the best separation and resolution of HMW proteins can be accomplished with a 3–8% Tris-acetate gel (Figure 1B). As seen in Figure 2, better transfer is seen using a Tris-acetate gel over a 4–20% Tris-glycine gel—9 ng visualized when a Tris-acetate gel was used vs 750 ng visualized when a Tris-glycine gradient gel was used in targeting ~190 kDA protein epidermal growth factor (EGFR).

Protein migration patterns on NuPAGE Bis-Tris, Novex Tris-Glycine, and NuPAGE Tris-Acetate gels
High molecular weight protein separation comparison on 4-20% Tris-Glycine, 3-8% Tris-acetate, and 8% Tris-Glycine gel

Figure 1. Tris-acetate gels afford the best separation of HMW proteins. A. Migration of different molecular weight proteins on select NuPAGE Bis-Tris, Novex Tris-glycine, and NuPAGE Tris-acetate gels. B. Comparison of HMW protein separation with HeLa cell lysate using different gel chemistries and gradients shows best separation of HMW proteins in the lysate using 3–8% Tris-acetate gels compared to commonly used gel types—4–20% Tris-glycine and 8% Tris-glycine. The top red line depicts where the stacking portion of the gel ends and the resolving portion of the gel begins. The second red line in each lane approximates how far a 200 kDa protein migrates into the resolving portion of the gel during electrophoresis.

Western blot detection of EGFR using a Tris-glycine and Tris-acetate gel for separation

Increased transfer efficiency and detection of HMW proteins is seen using a 3-8% Tris-acetate gel versus a gradient 4-20% Tris-glycine gel under the same experimental conditions.

Figure 2. Western blotting analysis of EGFR expression in A431 lysates transferred from an Novex 4–20% Tris-glycine gel and a NuPAGE 3–8% Tris-acetate gel using the iBlot 2 Gel Transfer Device. Method: Western blot analysis of EGFR was performed by loading serially diluted A431 cell lysate onto a NuPAGE 3–8% Tris-acetate gel (Cat. No. EA03752BOX) or onto a Novex 4–20% Tris-glycine gel, WedgeWell format (Cat. No. XP04202BOX). Proteins were transferred with the iBlot 2 Gel Transfer Device onto nitrocellulose membranes (Cat. No. IB23002) using the P0 program (10 min). Membranes were blocked for 30 minutes with Blocker FL Fluorescent Blocking Buffer (Cat. No. 37565) at room temperature and then probed overnight at 4°C with an EGFR polyclonal antibody (Cat. No. PA1-1110) at a dilution of 1:500. After overnight incubation, the membranes were washed in TBST and probed with Goat anti-Rabbit (H+L) Highly Cross-Adsorbed Secondary Antibody, conjugated to Alexa Fluor Plus 800 (Cat. No. A32735) at a dilution of 1:5,000 for one hour at room temperature.


Optimizing conditions for transfer of >150 kDa proteins

Recommendation 2: Increase transfer time

Rapid dry transfer with iBlot 2 Transfer device

The iBlot 2 Gel Transfer device has enabled many users to achieve rapid protein transfer for a broad range of proteins. The preprogrammed, default, 7-minute transfer protocol of the iBlot 2 device typically works well for a broad range of proteins. While these conditions work for many protein samples, parameters may need to be optimized for your protein(s) of interest and their respective molecular weights. Proteins >150 kDa migrate more slowly in a gel matrix relative to smaller molecular weight proteins and, as a result, require more time to transfer. For these HMW proteins, transfer times should be increased to 8–10 minutes regardless of the gel type selected. As demonstrated in Figure 3, the most efficient transfer of the ~190 kDa EGFR is achieved using transfer times of 8–10 minutes at 25 V.

Western blot detection of EGFR using the iBlot 2 transfer device with increasing transfer time
Relative band intensity in western blot detection of EGFR using the iBlot 2 transfer device at different transfer times

Figure 3. Effect of increasing transfer time on detection of EGFR using the iBlot 2 Gel Transfer Device. Method: Western blot analysis of EGFR was performed by loading serially diluted A431 cell lysate from 20 µg to 312 ng per well onto a Novex 4–20% Tris-glycine gel, WedgeWell format (Cat. No. XP04202BOX). Proteins were transferred onto nitrocellulose membranes via the iBlot 2 Gel Transfer Device (Cat. No. IB23002) at 25 V for 6 min, 7 min, 8 min, or 10 min, using the P0 program. Membranes were blocked for 30 minutes at room temperature with Blocker FL Fluorescent Blocking Buffer (Cat. No. 37565) and then probed overnight at 4°C with Invitrogen EGFR polyclonal antibody (Cat. No. PA1-1110) at a dilution of 1:500. After overnight incubation, the membranes were washed in TBST, probed with Invitrogen Goat anti-Rabbit (H+L) Highly Cross-Adsorbed Secondary Antibody, conjugated to Alexa Fluor Plus 800 (Cat. No. A32735) at a dilution of 1:5,000 for one hour at room temperature.


Recommended transfer parameters for proteins with molecular weights >150 kDa

Transfers stackMethodVoltageRun time
Regular transfer stack (Nitrocellulose or PVDF membrane- with one midi gel or two mini gels)P0, P320-258-10
Mini transfer stack (Nitrocellulose or PVDF- with one mini gel)P0, P320-258-10

If using a gel chemistry other than Tris-acetate, adding a quick alcohol equilibration step before transfer can greatly enhance the transfer of HMW proteins when not using the ideal gel chemistry. Equilibrating the gel in alcohol removes contaminating electrophoresis buffer salts and prevents an increase in the conductivity of the transfer, which can increase the amount of heat generated. In addition, the alcohol equilibration step allows the gel to adjust to its final size before transfer. Heat generated during the electrophoresis step can cause certain gels to expand; alcohol can help shrink the gel to its final size. To improve transfer efficiency, submerge the gel in 20% ethanol (prepared in deionized water), and equilibrate 5–10 minutes at room temperature on a shaker prior to transfer. Figure 3 demonstrates the increased transfer efficiency of keyhole limpet hemocyanin (KLH), a ~360–400 kDa protein, when the gel was equilibrated with 20% ethanol prior to transfer. Our data suggests that an equilibration step may not be needed with the Tris-acetate gels since large proteins from these gels transfer more efficiently than from Bis-Tris gels.

Western blot detection of KLH after 20% ethanol equilibration before transfer using the iBlot 2 transfer device

Increased transfer efficiency of KLH with pretreatment of a NuPAGE 4–12% Bis-Tris gel with 20% ethanol before transfer using the iBlot 2 transfer device.

Figure 4. Effects of an ethanol equilibrium step before transfer on the detection of KLH. Method: A dilution series of KLH was prepared for SDS-PAGE and loaded onto two Invitrogen Bolt 4–12% Bis-Tris Plus gels (NW04120BOX). Lane 1 was loaded with 5 µL of Invitrogen MagicMark XP Western Protein Standard (Cat. No. LC5602), lanes 2–10 were loaded with a 2-fold dilution series of KLH: 1,000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 3.9 pg, respectively, on each gel. After electrophoresis, one of the duplicate gels was incubated on a shaker in 20% ethanol for 10 minutes. The gels were then transferred using the iBlot 2 Gel Transfer Device to an iBlot 2 Transfer Stack, PVDF, mini (P0, 8 minutes). Immunodetection was performed on the two blots using the Invitrogen iBind Western Device (Cat. No. SLF1000) and associated Invitrogen iBind solutions (Cat. No. SLF1020) and Invitrogen iBind Cards (Cat. No. SLF1010). The primary antibody, anti-KLH, was diluted 1:2,000 in iBind solution. The secondary antibody, HRP conjugated goat anti-rabbit antibody, was diluted 1:1,000 in iBind solution. Upon completion of the 2.5-hour iBind reaction, blots were removed from the devices, briefly rinsed with deionized water, and then incubated in Invitrogen ECL Chemiluminescent Substrate (Cat. No. WP20005) for 5 minutes and imaged.


Rapid semi-dry transfer with Power Blotter Transfer device

Historically, semi-dry transfer systems have been considered ideal for mid- to low–molecular weight protein transfer, but suboptimal for HMW protein transfer. However, improvements to semi-dry transfer systems and better consumables have successfully enabled the transfer of HWM proteins using this technique. When using the Power Blotter Semi-dry System, proteins >150 kDa migrate more slowly and require more time to transfer. If your protein of interest is in this size range, it may be necessary to use a run time of 10-12 minutes for your transfer with 1-Step Transfer buffer. This high ionic strength transfer buffer allows for rapid transfers when paired with high-current power supplies, such as the Power Blotter. To directly compare the efficiency of HMW protein transfer using rapid semi-dry and wet transfer methods, western blot detection of three HMW protein targets, EGFR (190 kDa), mTOR (289 kDa), and Ecm29 (205 kDa), was performed using these methods in parallel (Figure 4). The gels were stained after the transfer process to examine the amount of protein left behind in the gel. The membranes were also stained using a reversible membrane stain and showed equivalent amounts of protein in each case. Western detection showed rapid semi-dry transfer using the Power Blotter system achieved equal or better transfer for HMW proteins targets versus wet transfer. These results demonstrate that using rapid semi-dry transfer methods for HMW protein transfer can save significant time without sacrificing western blot sensitivity or performance.

To further improve transfer efficiency using the Power Blotter system, we recommend using the Power Blotter Select Stacks. These pre-assembled transfer stacks contain a unique gel matrix technology used in the iBlot 2 transfer stacks. The gel matrices of the stack incorporate optimized anode and cathode buffers to act as ion reservoirs. The gel matrix allows the system to generate a high field strength that increases the transfer speed and efficiency. This format eliminates the need for pre-made buffers or soaking filter paper/membranes in transfer buffer, and minimizes handling that can lead to inconsistent performance.

Recommendations:

  1. Increase transfer time to 10–12 minutes
  2. Use ready-to-use Power Blotter Select Stacks

Figure 4. Rapid semi-dry transfer systems perform the same as or better than wet tank transfer for HMW protein transfer. HeLa lysates were prepared for SDS-PAGE and loaded on a NuPAGE 3–8% Tris-acetate gel. Lane 1 was loaded with 5 µL of PageRuler Plus Prestained Protein Ladder, lanes 2–11 were loaded with a dilution series of HeLa lysate, and lane 12 was loaded with 5 µL of HiMark Prestained Protein Standard. After electrophoresis, protein was transferred from gel to membrane using the described transfer technique and protocol. For the Power Blotter, membrane and filter paper were equilibrated for 5 minutes in 1-Step Transfer Buffer (Cat. No. PB7300). HMW program was selected for transfer at 10 minutes. For conventional tank transfer, 1 liter of Towbin transfer buffer was prepared with 20% ethanol and chilled to 4˚C. Filter paper and membrane were equilibrated in cold Towbin transfer buffer for 15 minutes. Gel was rinsed in deionized water and equilibrated in cold Towbin transfer buffer for 15 minutes. Transfer took place in a cold room for 2 hours at 25 volts constant. After transfer, the protein remaining in the gel was visualized using GelCode Blue Stain and the protein was transferred to the membrane with Pierce Reversible Protein Stain Kit. Membranes were imaged and then stain removed using the Stain Eraser component of the kit. The resulting membranes were rinsed in deionized water, cut into suitable strips, and the conventional western blot workflow was performed simultaneously on each. Membrane strips were blocked with Thermo Scientific Pierce Fast Blocking Buffer (Cat. No. 37575) for 5 minutes. Strips were incubated in Pierce Fast Blocking Buffer containing one of the following primary antibodies overnight at 4˚C: rabbit anti-EGFR Receptor PAb, 1:500; rabbit anti-mTOR PAb, 1:500; and rabbit anti-Ecm29 PAb, 1:200. Membranes were rinsed with Thermo Scientific Pierce Fast Wash Buffer (Cat. No. 37577) for 5 minutes and incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody, 0.25 ng/µL for 30 minutes at room temperature. Membrane strips were rinsed with ultrapure water and then washed five times for 5 minutes each in Pierce Fast Wash Buffer. Membranes were incubated in Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Cat. No. 34580) for 5 minutes and the chemiluminescent signal was imaged.

Wet tank transfer with Mini Gel Tank or SureLock Tandem Midi Gel Tank

Wet tank transfer traditionally has been the choice transfer technique for detection of high molecular weight proteins due to its flexibility. Transfer membranes of different pore sizes can be swapped easily, and transfer buffer formulations can be modified. This makes it easier to optimize transfer of high molecular weight proteins using less than ideal gel chemistries. When using Tris-acetate gels, highly efficient transfer of HMW proteins can be achieved using the recommended transfer protocols for the Mini Gel Tank and the SureLock Tandem Midi Gel Tank (Figure 5).

Recommended transfer parameters for proteins with molecular weights >150 kDa

Gel typeMembraneVoltageRun time
Mini Gel Tank with Mini Blot Module
NuPAGE 3-8% Tris-acetate (denatured)Nitrocellulose1060
PVDF2060
SureLock Tandem Midi Gel Tank with Midi Blot Module
NuPAGE 3-8% Tris-acetate (denatured)Nitrocellulose2530
PVDF2530

When using less than ideal gel chemistries, optimization of the transfer buffer may be needed. The addition of SDS (0.01–0.02%) to the transfer buffer can increase the mobility of proteins out of the gel and may help keep some higher molecular weight proteins in solution. At the same time, SDS can reduce protein binding to the membrane- specifically nitrocellulose due to the decreased hydrophobicity of the proteins. Alcohol has the opposite effect to SDS and may decrease protein mobility out of the gel. However, alcohol in the transfer buffer improves protein binding to nitrocellulose membranes by stripping the SDS from the proteins and increasing hydrophobic interactions with the membrane. Making modifications to the amount of alcohol in the transfer buffer, adding SDS, and increasing the transfer time can assist with the transfer efficiency of high molecular weight proteins.

Recommendations:

  1. Increase the transfer time by 5- or 10-minute increments
  2. Decrease the amount of methanol in the transfer buffer
  3. Add up to 0.02% SDS to the transfer buffer
  4. Use PVDF membranes
Western blot detection of high molecular weight protein BRCA2 after transfer using SureLock Tandem Midi Gel Tank

Figure 5. Chemiluminescent detection of BRCA2 (~380 kDa) protein transferred with the SureLock Tandem Midi Gel Tank. NuPAGE Tris-acetate 3-8% midi gel was loaded with decreasing amounts of HEK293 nuclear lysate and transferred onto a 0.45 PVDF membrane using the SureLock Tandem Midi Blot Modulefor 30 minutes at 25 volts. Membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer (Cat. No. 37565). For chemiluminescent detection, the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti BRCA2 (Cell Signaling Technology Cat. No. 10741, 1:500) and Rabbit anti-HDAC1 (Cat. No. PA1-860, 1:5,000,000) followed by an incubation with secondary antibody Donkey anti-Rabbit HRP (Cat. No. A16035, 1:5,000) in 1X Blocker FL for 1 hour. Membranes were incubated for 5 minutes with SuperSignal West Dura Extended Duration Substrate (Cat. No. 34075) and imaged on the iBright Imaging System.

Evaluating transfer efficiency

Regardless of transfer technique, there is often a large amount of protein remaining in the gel after transfer. Transfer efficiency is protein dependent and varies according to the size, abundance, charge, and hydrophobicity of each specific protein. Complete transfer of every protein is not possible, especially for abundant or overloaded proteins. If you stain the gel after transfer, you will find that some amount of protein remains in every gel type regardless of transfer technique. Nevertheless, there is typically sufficient protein on the membrane for subsequent western detection.

Prestained molecular weight protein ladders are considered to be an easy way to determine protein transfer efficiency. While prestained ladders can be used for monitoring protein transfer qualitatively (for example, correct orientation of the membrane and gel in the transfer sandwich), the extent to which they transfer to the membrane is an inaccurate indicator of actual protein transfer efficiency.

Prestained molecular weight protein ladders can often remain in the gel after transfer or can even migrate better than some samples proteins due to the dyes used. This does not indicate that the protein(s) of interest did not transfer fully. Prestained ladders contain chemically modified proteins. Covalent attachment of a hydrophobic dye to the ladder protein affects its charge, solubility, binding capacity, and transfer efficiency. As a result, the transfer efficiency of prestained ladders is not necessarily reflective of the transfer of your proteins of interest. Often, a portion of the HMW markers do not transfer out of high percentage non-gradient gels or gradient gels. This phenomenon is most notable in a 4–20% Tris-glycine gel.

Reversible membrane protein staining is a more reliable method of determining transfer efficiency. To monitor transfer efficiency, the membrane can be stained for total protein with a dye such as Ponceau S, amido black 10B, or Pierce Reversible Membrane Stain. Because dyes may interfere with antibody binding and detection, a protein stain that is easily removable is ideal.

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