An economical single-sided antibody incubation method for Western blotting

Abstract

A simple, single-sided antibody method for incubating primary and secondary antibodies in Western blotting was developed, which generates significant savings on the use of antibodies. Compared with the conventional immersion technique for antibody incubation, the present economical single-sided antibody incubation method resulted in a saving of 80% of antibody use. Besides, the present incubation method did not compromise the Western blot results and was not affected by the expression levels of target proteins.

Western blot (also known as protein immunoblot) is an analytical technique used for detection of specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate proteins, which are then transferred to a membrane where they are detected by probing with specific antibodies. The technique, allows determination of the approximate molecular weight of a protein and measurement of the relative amounts of the protein present in different samples (Renart et al., 1979; Towbin et al., 1979).

Western blotting is used widely in the fields of molecular biology, biochemistry, immunogenetics and other disciplines. Specific and high-quality antibodies are essential for Western blotting as they are able to bind specifically to a target protein in a mixture of thousands of proteins on a standard Western blot. Many antibodies are obtainable easily from commercial sources but remain relatively expensive and unaffordable for some laboratories. Any method which uses the minimum amount of antibody without compromising the result is of obvious utility. Incubating membranes in heat sealed bags/sealed plastic bags or applying reagents directly to one side of the membrane has been used to reduce the cost of expensive reagents (Dorri et al., 2009). However, using this method requires special bags and extra care to prevent folds in the membrane. The reagent runs out very easily and the membrane may dry when applying reagent to one side of the membrane. Microblots are now available which use just 100 µl or less of diluted anti sera (Canzler et al., 2009a; Canzler et al., 2009b). The procedure is much more complicated and it is especially useful for simultaneous protein detection of different antibodies when using limited samples. Although the unbound antibody can be reused between experiments, it is difficult to control the quantity and quality of reused antibody which may compromise the results.

In this study, an economical, simple, straightforward, single-sided antibody incubation method for Western blotting is described, which generates significant savings on the use of antibodies. The comparison of procedures of single-sided incubation and conventional immersion incubation is shown in Fig.1A.

Fig. 1. Single-sided antibody incubation and conventional immersion incubation.

(A) Detailed description of single-sided incubation (left side of the Figure) and conventional immersion incubation (right side of the Figure).

(B) Schematic view of Western blotting steps using single-sided incubation and conventional immersion incubation methods.

As proof-of-principle, expression of endogenous beta-actin in human embryonic kidney 293 cells (HEK-293) (Catalog No. CRL-1573, ATCC, Manassas, VA, USA), which are used commonly as transfection host cells, was tested. HEK-293 cells were collected by trypsinization, washed and pelleted by centrifugation. Cell pellets were lysed with RIPA buffer (150 mM sodium chloride, 1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5, and 2 mM EDTA) with protease inhibitors on ice for 30 min. After centrifugation, the supernatant was transferred to a new tube. Total protein concentration was measured using a Bio-Rad Protein Assay. Forty µg of the total protein lysate was mixed with an equal volume of Laemmli Sample Buffer Catalog No. 161-0737, Bio-Rad, Hercules, CA, USA) including 5 % beta-mercaptoethanol. Then the mixture was heated at 95 °C for 5 min and centrifuged at 20,800 × g for 5 min at 4 °C. The supernatant and 10 µl of BenchMark™ Pre-Stained Protein Ladder (Catalog No. 10748-010, Invitrogen, Carlsbad, CA, USA) were loaded into one well of a 15-well 4–20% Tris-HCl Ready Gel (Catalog No. 161-1123, Bio-Rad, Hercules, CA, USA). The gel was run at 100V for 30 min and then adjusted to 120V run for a further 90 min. A piece of polyvinylidene difluoride (PVDF) membrane was immersed for about 15 min in methanol, hydrated in deionized water then equilibrated in 1 × transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) until ready for use.

After the electrophoresis was completed the transfer “sandwich” was prepared and the protein was transferred at 20 V for 1 h using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Catalog No. 170-3940, Bio-Rad, Hercules, CA, USA). When completed, the side to which proteins were transferred was marked and the membrane was immersed in 30 ml of blocking buffer (5% non-fat dry milk in 1 × TBST buffer (0.1% Tween-20 in TBS)) overnight at 4 °C or 60 min at room temperature.

Next, anti-beta-actin mouse monoclonal antibody (Catalog No. sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was diluted to 0.1 µg/ml with non-fat milk blocking buffer (1% non-fat dry milk). The membranes were then incubated with diluted antibody using two methods. The conventional incubation method was to immerse the membrane in a small container with diluted antibody as shown in Fig.1A on the right. When using 85 mm × 60 mm membrane approximately 5 ml of diluted antibody was the minimum volume to cover fully the membrane. In the second method, a piece of parafilm, about 1.5 times the size of the PVDF membrane, was placed in an open container. Small drops of diluted antibody (10–15 µl per drop) were spaced evenly on the membrane (100–125 µl of diluted antibody or 10–12 drops per square inch) (Fig.1B on the left). The membrane was held using forceps with the marked protein side facing the antibody. It was then touched gently and slowly for about 10 seconds to the drops of antibody, starting at one side to prevent the formation of bubbles. The container was covered with a lid to prevent evaporation and the membrane was incubated overnight at 4 °C. After washing 3 times with 1× TBST buffer, the membranes were incubated with 1/2000 diluted horseradish peroxidase (HRP)-conjuncted goat anti-mouse secondary antibody (Catalog No. 1858413, Pierce, Rockford, IL, USA) for one hour at room temperature. Two different incubation methods were used as described previously.

The bound antibodies were visualized by enhanced chemiluminescence (Western blotting Luminol Reagent: Catalog No. sc-2048, Santa Cruz Biotechnology, Santa Cruz, CA, USA) again using the two different incubation methods. Finally, target proteins were detected with an LAS-3000 imaging system.

Fig.2A shows a comparison of beta-actin bands generated using the single-sided incubation method (Fig.2A on the left) and the conventional incubation method (Fig.2A on the right). Clearly, the single-sided, economical method performed as well as the conventional method.

Fig.2. Western blot analysis of different proteins using single-sided incubation.

(A) Detection of beta-actin in HEK-293 cells using the single-sided antibody incubation method (left side of the Figure) and conventional immersion incubation method (right side of the Figure). Sample loading was duplicated. Primary antibody, 0.1 µg/ml beta-actin (C4) antibody (sc-47778).

(B) Western blot analysis of recombinant proteins expressed in E.coli using the single-sided antibody incubation method. “−” and “+” indicate non-induced and IPTG induced expressions, respectively. Primary antibody, 0.1 µg/ml His Probe (H-15) antibody (sc-803).

(C) Various concentrations of TAT fusion proteins were added to the culture medium of IMR90 cells and incubated for 8 hours, and the presence of the proteins which entered into the cells, was examined by Western blotting using the single-sided incubation method and detected by 0.1 µg/ml specific antibodies.

This method was then examined to determine whether it can be used to detect exogenous gene expression in transformed cells and transduced cells. The expression of target recombinant proteins (his-TAT-c-Myc, his-TAT-Oct4 and his-TAT-Sox2) was produced by plasmid constructs transformed in E.coli and induced with 1 mM IPTG. Ten µg of protein lysates from non-induced and IPTG-induced transformed cells were analyzed by Western blotting using His probe antibody (Catalog No. Sc-803, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as the primary antibody (Fig.2B). Various concentrations of purified recombinant proteins were added to the culture medium of IMR90 fibroblasts cells in a 24-well tissue culture plate. After 8 h of incubation, the whole well of cells was collected, and the presence of the proteins which entered into the cells with the help of the TAT membrane penetrating motif, was examined by Western blotting (Fig.2C) using specific antibodies (anti-c-Myc antibody: Sc-40; anti-Oct3/4 antibody: Sc-5279; anti-Sox2 antibody: Sc-20088; Santa Cruz Biotechnology, Santa Cruz, CA, USA). All of these primary antibodies were diluted to 0.1 µg/ml, which represented the optimal concentration used in standard Western blotting techniques. The data clearly show that this single-sided, economical method can be used to detect exogenous gene expression.

In conclusion, the single sided incubation method did not compromise the sensitivity of Western blot providing results equivalent to those using more conventional methods of antibody incubation.

The main advantage of this economical incubation method is a saving of approximately 80% of primary and secondary antibodies and Western blotting luminol reagent. Although only luminol detection was carried out in this study due to its high sensitivity, reagent savings would also apply when using colorimetric assays which use DAB or TMB. In other words, the amount of antibodies and reagents consumed normally by one blot can now produce results from five blots, which is a significant saving in Western blot analysis.