Reliable transfer of high molecular weight (HMW) proteins (i.e., >200 kDa) from a gel to membrane during western blotting is a common challenge among life science researchers. 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. We compared results between different methods, specifically wet tank and rapid semi-dry transfer.

In traditional wet tank transfer, or electroblotting, the transfer sandwich is secured in a cassette and submerged in a tank filled with transfer buffer. Typically, tank transfer uses a large volume of transfer buffer containing methanol or ethanol and is usually performed with an ice pack inside the transfer tank or by placing the whole apparatus in a cold room to offset the heat that is generated. Tank transfer can take from 1 hour to overnight to complete.

With semi-dry transfer, the gel and membrane are sandwiched between two pieces of filter paper that have been saturated in transfer buffer. The semi-dry technique has become quite popular because it uses significantly less transfer buffer and generally requires 30 to 60 minutes to complete. More recently, rapid dry or semi-dry transfer systems have been commercialized to further decrease the transfer time required to 7 to 10 minutes. Two such systems are the Invitrogen™ iBlot™ 2 Gel Transfer Device (Cat. No. IB21001) and the Thermo Scientific™ Pierce™ Power Blotter (Cat. No. 22834). 


Using the right gel for HMW protein electrophoresis

When targeting HMW proteins for your transfer, it is best to use a Tris-acetate gel, or low percentage Tris-glycine gel. For example, 4–20% Tris-glycine gradient gels are very popular for electrophoresis because of their ability to separate a very broad range of proteins (20 to 200 kDa). However, they are not recommended for immunodetection of HMW proteins because proteins >200 kDa are compacted into a very narrow region at the top of the running portion of the gel. For immunodetection of proteins larger than 200 kDa use a Tris-acetate gel or low percentage Tris-glycine gel (Figure 1). 


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Figure 1. Tris-acetate gels afford the best separation of HMW proteins. Images depict different gel types stained using Thermo Scientific™ Pierce™ Power Stainer (Cat. No. 22833) post-electrophoresis. A. Invitrogen™ Novex™ Wedgewell™ 4–20% Tris-Glycine Mini Gel (Cat. No. XP04200BOX). The lanes containing Thermo Scientific™ PageRuler™ Unstained Protein Ladder (Cat. No. 26614) are highlighted to show condensed HMW proteins at top of gel. B. Invitrogen™ NuPAGE™ Novex™ 3–8% Tris-Acetate Protein Gel (Cat. No. EA0375BOX). HMW proteins are further spread and thus easier to uniquely identify versus 4–20% Tris-glycine gels. C. 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. The top red line depicts where the stacking portion of the gel ends and the separating portion of the gel begins. The second red line in each lane approximates how far a 200 kDa protein migrates into the separating portion of the gel during electrophoresis.


Gel staining does not reflect protein 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. The gel is usually at least 10 times thicker than the blotting membrane, and complete transfer of every protein is not possible, especially for abundant or overloaded proteins (Figure 2). If you stain the gel after transfer you will find that some amount of protein remains in every gel type regardless of transfer technique (Figure 3). Nevertheless, there is typically sufficient protein on the membrane for subsequent western detection (Figures 4 and 5).


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Figure 2. Rendering of Pierce™ Power Blotter set up for rapid transfer. Gel thickness depicted in relation to membrane capacity. The extent of protein binding to the membrane is protein-dependent, varying according to size, abundance, charge, and hydrophobicity of each specific protein.

Reversible membrane stain as a method to assess protein transfer efficiency

Prestained molecular weight 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.

Often times residual prestained molecular weight protein ladders will remain in the gel after transfer. 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 higher molecular weight markers do not transfer out of a high percentage non-gradient gel or gradient gel. This phenomenon is most notable in a 4–20% Tris-glycine gel (Figure 3). It is important to use the correct amount of prestained molecular weight markers so as to not overload the gel, resulting in incomplete transfer of the prestained markers.

Reversible protein staining is a more reliable method of determining transfer efficiency (Figure 3). Even though there seems to be differing amounts of protein in the gel after the transfer process, the reversible staining of the membrane shows relatively similar amounts of protein transferred to the nitrocellulose membrane regardless of transfer technique. As seen in the gels stained after transfer, prestained proteins exhibit different transfer efficiency than unstained native proteins. The amount of unstained ladders that remain in the gel after transfer is similar for all transfer techniques.


Figure 3 . Titrations of the indicated molecular weight ladders were electrophoresed on Novex 4–20% Tris-glycine gels. Lanes 1–4 contain Thermo Scientific™ PageRuler™ Plus Prestained Protein Ladder (10 to 250 kDa; Cat. No. 26620), 2.5 µL, 5 µL, 10 µL, and 15 µL, respectively. Lanes 5–8 contain Thermo Scientific™ HiMark™ Prestained Protein Standard (31 to 460 kDa; Cat. No. LC5699), 5 µL, 10 µL, 15 µL, and 20 µL, respectively. Lanes 9–12 contain PageRuler Unstained Protein Ladder (10 to 200 kDa), 2.5 µL, 5 µL, 10 µL, and 15 µL, respectively. After electrophoresis, the protein ladders were transferred to a nitrocellulose membrane using the Pierce Power Blotter or conventional tank transfer technique. The gels and membranes were stained before and after transfer using the Pierce Power Stainer for the gels before transfer, Thermo Scientific™ GelCode™ Blue Stain Reagent (Cat. No. 24590) for the gels after transfer, and Thermo Scientific™ Pierce™ Reversible Protein Stain Kit (Cat. No. 24580) for visualizing the amount of protein that was transferred to the membrane.

Rapid semi-dry transfer methods can be used for western blotting of HMW proteins.

Historically, semi-dry transfer systems have been considered ideal for mid- to low–molecular weight protein transfer but suboptimal for HMW protein transfer. Wet tank transfer, on the other hand, is often considered the optimal method for HMW protein transfer. To directly compare the efficiency of HMW protein transfer of rapid semi-dry and wet tank 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 the rapid semi-dry transfer systems performed the same as or better than wet tank transfer for HMW proteins. 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. 


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 50, 10, 5, 2.5, 1.0, 0.5, 0.25, 0.10, 0.05, and 0.02 µg of HeLa lysate (protein load), respectively, 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 Pierce Power Blotter, membrane and filter paper were equilibrated for 5 minutes in Thermo Scientific™ Pierce™ 1-Step Transfer Buffer (Cat. No. 84742 ). The gel and membrane were sandwiched between filter paper and orientated inside the Thermo Scientific™ Pierce™ Power Blot Cassette (Cat. No. 22835 ) with membrane closest to the anode (bottom plate). The Power Blot Cassette was placed into the Thermo Scientific™ Pierce™ Power Station (Cat. No. 22838 ) and the HMW program was selected for transfer in less than 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. The equilibrated gel and membrane were sandwiched between filter paper, secured into a blotting cassette and orientated inside of a blotting tank with membrane closest to anode. 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 transferred to 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 conventional western blot workflow 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. 34080)  for 5 minutes and the chemiluminescent signal was imaged on the Thermo Scientific™ myECL™ Imager (Cat. No. 62236). 

 


To further demonstrate the performance of rapid semi-dry transfer of HMW proteins, western blots were performed to detect Nestin from neuronal stem cells comparing different transfer methods (Figure 5). Nestin, 240 kDa, is an intermediate filament protein expressed mostly in nerve cells that is implicated in the radial growth of the axon. Western blot detection using rapid semi-dry transfer was equivalent or better than wet tank transfer even though prestained protein ladders partially remain in the gel.


Figure 5: Detection of Nestin in neuronal stem cells using NuPAGE 3–8% Tris-acetate gels. Nitrocellulose membranes were used for western blotting. First two lanes in each gel include PageRuler Prestained Protein Ladder and HiMark Prestained Protein Standard, respectively. A. Two gels served as controls, where one was stained using the Pierce Power Stainer and the other using Thermo Scientific™ Imperial™ Protein Stain (Cat. No. 24615 ). B. Three other gels were transferred using either rapid semi-dry (Pierce Power Blotter, preprogrammed method for HMW proteins with 10 minute transfer) or wet tank transfer (Towbin buffer for 2 hours at 0.25A) methods. C. The gels were stained after transfer using Imperial Protein Stain to show any remaining protein in the gels. D. The membranes were imaged after being reversibly stained using Pierce Reversible Protein Stain and then processed for immunodetection. E. Nestin was detected using mouse anti-Nestin monoclonal antibody (clone 10C2) , horseradish peroxidase–conjugated goat anti-mouse secondary antibody followed by Thermo Scientific™ SuperSignal™ West Dura Extended Duration Substrate (Cat. No. 34075), and the blots were imaged using the myECL Imager.


Summary

The study of HMW proteins is an increasingly important area of life science research. Historically, the preferred method to enable western blotting of proteins >200 kDa has been wet tank transfer. The more recent development of rapid semi-dry transfer methods has greatly reduced the time required for the western blotting workflow. The results of this study indicate that the detection of HMW proteins can be successfully performed using rapid semi-dry transfer, which offers a viable alternative to traditional time-consuming methods. Additionally, it’s critical to select the appropriate gel type and staining method for both optimizing and determining protein transfer efficiency during western blotting.



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Pierce Power Blotter

The Thermo Scientific™ Pierce™ Power Blotter is designed specifically for rapid semi-dry transfer of 10–300 kDa proteins from polyacrylamide gels to nitrocellulose or PVDF membranes in 5 to 10 minutes. The Pierce Power Blotter features an integrated power supply optimized to enable consistent, high-efficiency protein transfer when used with commonly used precast or homemade gels (SDS-PAGE) and nitrocellulose or PVDF membranes.

Features of the Pierce Power Blotter include:

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