Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting

Abstract

SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin’s transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

INTRODUCTION

SDS-PAGE is one of the most commonly used methods in protein biochemistry laboratories for the analysis of crude and purified protein samples. Separation of proteins by electrophoresis was originally developed by Ornstein and Davis,¹ where serum proteins were resolved in the absence of denaturing and reducing agents. This type of electrophoresis can preserve the native charge of protein molecules but is not suitable for resolving the subunits and their MWs. To overcome these limitations, Laemmli² incorporated the detergent SDS to the protein gel electrophoresis methods. SDS-PAGE is now the most popular form of protein gel electrophoresis. The SDS, assisted by boiling and reducing agents, such as DTT and 2-ME, breaks down the disulfide bonds, destroys the tertiary structure, and linearizes the protein molecules. SDS also coats protein with its negative charge fairly uniformly, making the charge of protein approximately proportional to its MW; as a result, it migrates toward a positively charged electrode (anode), based on the molecular mass of proteins. In SDS-PAGE, acrylamide serves as a size-selective sieving matrix, allowing small-size protein molecules to move faster than the large-size protein molecules. This property of acrylamide allows its concentration as a determining factor in the speed of migration of proteins based on their mass. In other words, small-size protein molecules can be resolved in the presence of higher concentrations of acrylamide and vice versa. To accommodate a wide range of proteins differing in their mass, gradient gels can be used.³ Proteins separated based on the MWs can be visualized by a variety of stains. The most popular among them are Coomassie blue and Ponceau-S stains.

Protein molecules separated by SDS-PAGE are then transferred to solid surfaces; most common among them are microporous membranes, such as nitrocellulose and PVDF. Protein specific band(s) on these membranes are visualized by a procedure called immunoblotting or Western blotting.⁴ Buffers for SDS-PAGE and Western blotting are crucial in separation of proteins and their subsequent transfer to membranes. Tris-glycine-SDS is the buffer of choice by many. The only difference between the gel EB and TTB, however, is that the latter contains 10–20% methanol. A toxic substance,⁵ methanol serves in removing SDS from the protein bands, facilitating their binding with the membrane, and preventing swelling of the gel during transfer.⁶ The reuse of TTB in Western blotting, primarily to reduce toxic waste without compromising the quality of the Western blots, was reported earlier.⁷ Dorri et al.,⁸ in their commentary, later disagreed with this claim, however, without providing any supporting data.

Therefore, the purpose of this paper is to reanalyze the multiple use of TTB and support our findings with actual data. Additionally, we present data on the effects of multiple use of EB on protein profiles as well on the quality of subsequent Western blotting of a group of proteins from low to high MW range in the commercially obtained protein MW standards. We have also investigated the effects of reusing EB and TTB on immunoblotting of a high MW protein, CFTR (molecular mass ∼170 kDa). Our laboratory routinely performs CFTR Western blotting for various ongoing research projects. CFTR functions as a chloride ion channel on the plasma membrane of many epithelial cells. A mutated CFTR is the cause of genetic disease cystic fibrosis.⁹

MATERIALS AND METHODS

Materials

Precast, 4–20% Mini-PROTEAN TGX gels, Immuno-Blot PVDF membrane (pore size 0.2 μm), Tris/glycine buffer, Precision Plus Protein WesternC MW standard, Strep Tactin, and all other routine chemicals required for SDS-PAGE and Western blotting were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Novex sharp, unstained MW standard and Halt protease inhibitor cocktail were purchased from Thermo Fisher Scientific Life Sciences (Waltham, MA, USA). AcquaStain protein gel stain was purchased from Bulldog Bio (Portsmouth, NH, USA). BSA (IgG-free) and goat anti-rabbit horseradish peroxidase (HRP) secondary antibody were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). General laboratory chemicals and reagents for tissue culture were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and/or from Thermo Fisher Scientific. FBS was purchased from Atlanta Biologicals (Lawrenceville, GA, USA).

Cell Lysates

Cystic fibrosis bronchial epithelial (CFBE)41₀, a human lung epithelial cell line transfected with the wild-type CFTR gene (CFBE-wt), as originally developed by Dr. J. P. Clancy (University of Cincinnati, OH, USA),¹⁰ was generously provided by Dr. Bruce Stanton (Geisel School of Medicine at Dartmouth, Hanover, NH, USA). These cells were grown in a humidified atmosphere at 37°C in the presence of 5% CO₂ in the following: DMEM with 2 mM l-glutamine, 35 mM sodium bicarbonate, penicillin (50 U/ml), streptomycin (50 μg/ml), plasmocin (5 μg/ml), puromycin (0.5 μg/ml), and 10% FBS. CFTR expression in these cell lines was stimulated as described previously.¹¹ Following stimulation, cells were lysed with 1% SDS containing Halt protease and phosphatase inhibitor cocktail.

Protein Assay

Cell lysates were assayed for total protein content using the improved Amidoschwarz protein assay.¹²

SDS-PAGE

Known quantitates of protein samples were electrophoresed using a precast, 4–20% Mini-PROTEAN TGX gels on a Mini-PRETEAN Tetra cell (Bio-Rad Laboratories), as per the standard procedures² and manufacturer’s instructions. The gel EB (25 mM Tris, 192 mM glycine, 0.01% SDS) was used in excess (∼50 ml) for the first electrophoretic run. This extra volume can be easily accommodated by the Mini-PROTEAN Tetra cell apparatus. Special care was taken to recover the maximum volume of buffer after each run for it to be reused in the subsequent runs. No more than ∼10 ml buffer after each run and no more than ∼50 ml buffer after the fifth and final run were lost. Protein bands were visualized by staining with a Coomassie-based AcquaStain protein gel stain. All gels were destained with deionized water twice for 30 min, followed by overnight destaining on an Orbit LS low-speed laboratory shaker (Labnet International, Edison, NJ, USA). Digital images of destained gels were captured using a standard desktop scanner (Epson Perfection V33) and either saved as or converted to tagged image file format (TIFF). Selected protein bands representing a wide range of molecular mass (viz., 10, 50, 260 kDa) were quantified using Quantity One 1-D analysis software (Bio-Rad Laboratories). Precise square boxes of the same size were drawn around each protein band. Density volume within each box was measured as intensity/mm². Average of density volume from 3 independently run gels was used to calculate sem. All data were subjected to a two-way ANOVA to determine the significance of difference between protein bands from Gel #1 (fresh EB) to Gel #6 (5× used EB).

Western Blotting

Following electrophoresis, gels were transferred to a PVDF membrane with TTB (25 mM Tris, 192 mM glycine, 20% methanol, 0.01% SDS, pH 8.3) using the Mini Trans-Blot electrophoretic transfer cell (Bio-Rad Laboratories). Similar to gel EB, TTB was also carefully collected to minimize its loss after each transfer so that it can be reused for the subsequent electrotransfers. When necessary, the lost buffer volumes were replaced by dropping a prefrozen polystyrene test tube containing buffer or frozen ice pack into the buffer tank. Afterwards, protein transfer membranes were blocked with a 5% blotto (nonfat dry milk powder). Membranes with the WesternC MW standard were incubated with 1:10,000 dilution of goat anti-rabbit secondary antibody containing Strep Tactin, as per the manufacturer’s instructions. Western blotting procedure for the detection of CFTR protein was carried out, as per the previously established procedures, using rabbit polyclonal anti-CFTR antibody R3194.^{12, 13} Protein-specific signals were detected by chemiluminescence with the help of Clarity Western ECL substrate on the ChemiDoc image analyzer (Bio-Rad Laboratories). Protein-specific bands from the digital images were scanned and analyzed, as per the procedure described above for SDS-PAGE.

RESULTS AND DISCUSSION

The reuse of gel EB and TTB in Western blotting is not only economical, in terms of time invested in their preparation and cost involved, but also helps to reduce the toxic waste, especially in the case of TTB, as a result of the presence of methanol. Of these, time and cost factors may be of particular importance to small, undergraduate research laboratories such as ours. Two previous publications^{7, 8} have presented the opposing views on the effects of reusing of TTB on Western blotting. Reuse of methanol, suggested by Pettegrew et al.,⁷ was mainly to reduce the toxic waste generated by methanol that is normally present at 10–20% levels in TTB. Methanol, when entering the body through ingestion, inhalation, or absorption through skin in quantities as low as 10 ml, may lead to blindness by damaging optic nerves.⁵ In this paper, we have investigated not only the effects of reusing of TTB but also of reusing EB for at least up to 5 times on a Western blotting signal of various proteins, ranging from low to high MW range.

We have run a total of 6 gels and blots, where the first gel and blot (i.e., #1) received the fresh buffers, and the last gel and blot (i.e., #6) received the 5× used buffers. One of the challenges for conducting this type of experiment was to minimize the loss of buffer volumes from 1 run to the next to avoid adding fresh buffer each time. We did lose a volume of ∼10 ml EB during each SDS-PAGE run, resulting in ∼50 ml total EB loss by the last and sixth SDS-PAGE run. To counter this loss, we started the first electrophoretic run by adding ∼50 ml EB in excess, which could easily be accommodated by the buffer tank of the Mini-PROTEAN Tetra cell. Likewise, we encountered a loss of ∼20 ml TTB each time during the second and third electrotransfers. Replacement of these lost volumes with fresh TTB was unnecessary, as electrode wires of the Mini Trans-Blot apparatus still remained submerged into the TTB. During the fourth and fifth transfer, however, when we noticed the fall of the TTB level slightly below the electrode wire assembly, a prefrozen ice pack or polystyrene test tube containing TTB was dropped into the buffer tank to raise the level. This practice allowed us to avoid adding any fresh buffer during this study.

Effects of Reusing EB on SDS-PAGE Protein Profiles

First, we wanted to determine the effects of reusing EB on electrophoretic separation of a MW standard, consisting of proteins with molecular masses ranging from 10 to 260 kDa. We did not notice any visible impact of reusing the same EB for at least up to 5× on protein profiles of this standard (Fig. 1A). Three of the protein bands representing low (10 kDa), medium (50 kDa), and high molecular masses (260 kDa) were scanned, as per the procedure described in Materials and Methods. Our data suggest a nonsignificant effect of reusing EB on the density of these protein bands (measured as intensity/mm²), based on a two-way ANOVA from Gel #1 (i.e., fresh EB) to Gel #6 (i.e., 5× used EB; Fig. 1B).

FIGURE 1.

A) Effects of reusing gel EB on a protein profile. A 5 μl Novex sharp, unstained, wide-range protein a MW marker was electrophoresed on individual, 4–20% Mini-PROTEAN TGX gels (Gel #1–6). Fresh EB was used for Gel #1. EB used for Gel #1 was saved and reused for the next gel (Gel #2, i.e., 1 time used). Likewise, after each run, the EB was saved and reused for all subsequent gels, where Gel #6 received 5 times used EB. Proteins were stained, destained, and imaged, as per the procedures described in Materials and Methods. Each of the lanes in this figure is excised from an individual gel and put together for comparative analysis. These gels represent 1 of the 3 sets of gels that were electrophoresed. Protein bands marked by arrows on the right-hand side were scanned, and their density was measured using Quantity One 1-D analysis software (see B). B) Effects of reusing gel EB on representative protein bands. Density of electrophoretic bands representing low (10 kDa), medium (50 kDa), and high (260 kDa) molecular mass in A was measured as intensity/mm² using Quantity One 1-D analysis software. Densities from 3 independent sets of experiments were averaged, and sem was calculated and plotted. Band intensities of these bands remain unchanged from freshly used EB (Gel #1) to 5 times used EB (Gel #6), as determined by a two-way ANOVA.

We also analyzed the effects of reusing EB on a purified protein BSA, as well as crude cell lysate prepared from CFBE-wt cells. Like the protein MW standard, we did not see any negative effect of reusing EB on electrophoretic resolution of these later samples (data not shown).

Effects of Fresh and Used EB and TTB on Western Blots

Next, we wanted to analyze what happens when both EB and TTB are reused in Western blot experiments. As anticipated by Dorri et al.⁸ in their commentary, we clearly noticed a decline in signal of all proteins in the range of 10–250 kDa in a WesternC protein standard (Fig. 2). Commercially purchased WesternC protein standard is a mixture of 10 recombinant proteins (10–250 kDa) and affinity tagged with Strep-tag peptides.¹⁴ These peptides can strongly bind to the native form of streptavidin, as well as its genetically modified form, called Strep-Tactin. With the use of this chemical interaction that is similar to the well-known, irreversible, avidin-biotin-binding,¹⁵ Strep-tagged proteins in WesternC, standards can be determined by using Strep Tactin in Western blotting, as described in Materials and Methods.

FIGURE 2.

A) Effects of reusing gel EB and TTB on Western blotting of proteins of different MWs. A 7.5 μl Precision Plus Protein WesternC MW standards were electrophoresed on an individual, 4–20% Mini-PROTEAN TGX gel and immunoblotted with goat anti-rabbit HRP-conjugated secondary antibody containing Strep Tactin, as per the manufacturer’s instructions. Strep Tactin bound to the Strep-tagged WesternC protein standard was detected by chemiluminescence (see Materials and Methods). Western blot (WB) #1 received fresh EB and TTB. Both of these buffers were reused for subsequent Western blot experiments, as described in Fig. 1A. Protein bands, marked by the arrow on the left-hand side, were scanned, and their density was measured using Quantity One 1-D analysis software (see B). Reuse of EB and TTB produced a demising signal in Western blots for all proteins regardless of MW. A persistent signal for a 20 kDa size protein may be a result of its strong, initial intensity compared with the rest of the proteins. Each of the lanes in this figure is excised from an individual Western blot image and pooled together for comparative analysis. These Western blot images represent 1 of the 5 sets of independent experiments. B) Effects of reusing EB and TTB on representative protein bands in Western blotting. The density of electrophoretic bands representing low (10 and 20 kDa), medium (50 kDa), and high (250 kDa) molecular masses in A was measured as intensity/mm² using Quantity One 1-D analysis software. Densities from 5 independent sets of experiments were averaged, and sem was calculated and plotted. All of the proteins analyzed showed a reduction in Western blot signal when EB and TTB are reused.

A signal of all protein components of the WesternC protein standard either completely disappeared with the first reuse of TTB in the case of the 25 kDa size protein or is reduced in the case of the rest of the other proteins (Fig. 2A). A further decline in signal with the continuous reuse of TTB, however, was relatively less striking (see Lanes 3–6 in Fig. 2A). A signal of 1 of the proteins in this mixture (20 kDa), after its initial decline with first reuse of TTB, remained relatively steady in the rest of the Western blots. Part of the reason for this steady signal could be a result of high initial intensity of this particular protein in the standard mix. Effects of reuse of EB and TTB were analyzed further on a high MW membrane protein CFTR (molecular mass ∼170 kDa), with which we routinely work within our laboratory. Like various proteins in the WesternC standard, the CFTR protein-specific signal was also declined with continuous reuse of TTB (Fig. 3). As reuse of EB had no effect on the protein profiles (Fig. 1), we obviously wanted to check what happens when EB buffer is reused, and TTB is used fresh each time. As expected, we did not see any decline in a CFTR-specific protein signal in this latter case (Fig. 4). Similar observations were found with a low MW trafficking protein Rab11 (molecular mass ∼25 kDa; data not shown).

FIGURE 3.

Effects of reusing gel EB and TTB on Western blots. A 25 μg CFBE-wt cell lysate was electrophoresed on individual, 4–20% Mini-PROTEAN TGX gels, as described in Fig. 1, transferred to PVDF membrane, and subjected to Western blotting. Fresh EB and TTB were used for Gel/Blot #1. EB and TTB used for Gel/Blot #1 were saved and reused for the next gel/blot (#2; 1 time used). Likewise, after each run, the EB and TTB were saved and reused for all subsequent gels/blots, where Gel/Blot #6 received 5 times used EB and TTB. A CFTR-specific protein band (molecular mass ∼170 kDa) was detected by chemiluminescence on the ChemiDoc image analyzer. A WesternC MW marker (MWM) is shown on the left-hand side. A reduction in the CFTR-specific signal was noticed with the continuous reuse of buffers. Each of the lanes in this figure is excised from an individual Western blot image and put together for comparative analysis.

FIGURE 4.

Effects of reusing gel EB and fresh TTB on Western blots. A 25 μg CFBE-wt cell lysate was electrophoresed on individual, 4–20% Mini-PROTEAN TGX gels, transferred to a PVDF membrane, and subjected to Western blotting. Fresh SDS-PAGE buffer (EB) was used for Gel #1. EB used for Gel #1 was saved and used for the next gel, as described in Fig. 1. TTB, however, was used fresh each time, unlike for experiments described in Fig. 3. CFTR-specific protein band (molecular mass ∼170 kDa) was detected by chemiluminescence on the ChemiDoc image analyzer. The WesternC MW marker is shown on the right-hand side. A CFTR-specific signal remained steady up to Gel #6, with continuous reuse of EB (i.e., 5 times) and fresh TTB each time. Each of the lanes in this figure is excised from an individual Western blot image and put together for comparative analysis.

We believe that methanol loses its effectiveness in terms of its capacity to help bind the proteins to the membrane as a result of its oxygenation and evaporation with continuous use. Methanol is known to undergo oxygenation in open air and converts into carbon dioxide based on a well-known combustion reaction.¹⁶ Alternative conditions, such as a reduction to no methanol in TTB, are recommended for high MW proteins.¹⁷ In our experience, however, methanol, lower than 20% under the given condition, resulted in a lesser-than-optimal signal for CFTR protein (data not shown). Ethanol, a less-toxic substance than methanol, presumably can also be used. However, cost and risk of having ethanol in the laboratory as a result of regulations by TTB (Alcohol and Tobacco Tax and Trade Bureau), are involved. The ionic strength of TTB is also a determining factor on blotting time and efficiency. TTB with lower ionic strength may result in reduced blotting speed.^{18, 19} Hence, the depletion of the ionic strength of TTB, with its reuse supplemented with loss of SDS, may have negative effects on proteins transfer to membranes and ultimately, their detection by Western blotting.

Based on our analysis reported in this paper, it is perfectly fine to reuse EB for multiple times (at least up to 5×) without compromising the overall quality of protein profiles and subsequent Western blotting. TTB, on the other hand, is not recommended for reuse, as a result of the decline in the protein-specific signal in Western blotting procedures regardless of MW size.