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Western blot

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Western blot is a powerful technique in that; it leads to simultaneously detection of a specific protein by means of its antigenicity and its molecular mass. The proteins are first separated by mass using , transferred from gels onto membranes and then specifically detected in the immunoassay step. Information on activation status (i.e. enzyme pro-form vs. active species, unless specific antibodies against the pro species are available), oligomeric arrangement and post-translational modifications can also be deduced from this technique.

The efficiency of protein transfer from the SDS-PAGE gel to the support membranes can be affected by the gel thickness and the total acrylamide concentration. Transfer buffers can also affect the efficiency of protein migration through the gel. While methanol is necessary to prevent gel swelling with heating, and to keep protein adsorbed to membrane, methanol-containing buffers often result in the decrease in pore size and may even precipitate proteins within the gel. Poor transfer can be remedied by using methanol-free transfer buffer.

The choice of support membranes for protein transfer is usually determined primarily by the subsequent investigation steps to be performed. For conventional probing with antibodies, nitrocellulose (high binding capacity ~80-100 μg/cm2) or nylon (binding capacity ~480 μg/cm2) are suitable. Although nylon has an attractive binding capacity, blocking steps are usually performed at high temperatures. This may denature sensitive antigens and these membranes do not stain well with the anionic dyes required to check protein transfer efficiency.

In western blot, the transfer of proteins in membranes is mostly followed by immunoassay step where specific antibody is used to probe for specific antigens. Immunoassay involves the following steps: Blocking; where the transfer membranes are blocked with a concentrated protein solution (e.g. ovalbumin, BSA, haemoglobin, non-fat milk powder) to prevent further non-specific binding of proteins [non-protein (polyvinylpyrrolidone) in the presence or absence of non-ionic detergent (such as Tween 20), may also be used for blocking]. This step is followed by incubation of the membrane in a diluted antibody solution, washing of the membrane, incubation in diluted conjugated probe antibody or other detecting reagent, further washing, and finally detection.

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1. Transfer of protein to the membrane

Apparatus and equipment

  • Transfer cassette: Hoefer® TE series Transphor Electrophoresis Unit
  • HybondTM -C nitrocellulose paper (0.45 μm pore size) supplied as 33 x 300 cm rolls
  • Filter paper: Whatman 3mm or preferably blotting paper. Both nitrocellulose paper and filter paper are cut to the same size as gel using clean Blade and gloves.
  • Blotting Electrodes are carbon slabs, 1 cm thick x 20 cm long x 15 cm wide, with heavy insulated leads, or patent blotting apparatus.
  • Powerpack: Normally a high-amperage but low-voltage apparatus is preferred; one that is capable of supplying 500 mA – 1 A current at voltages as low as 5 V. Paul Meyers electroblotting apparatus is suitable.

Reagents

  • Gershoni blotting buffer [25 mM Tris-HCl, 192 mM glycine, 20% (v/v) methanol, 0.01% (w/v) SDS, pH 8.3]. 6.05 Tris, 28.8 g and 2 ml of 10% (w/v) SDS solution are dissolved in 1.6 litres dd.H2O. 400 ml Methanol is added and the solution is stored at -20°C without pH adjustment.
  • Ponceau S proteins stain solution [0.1% (w/v) in 1% (v/v) acetic acid]. 0.1 g Ponceau S and 1 ml acetic acid are added to a 100 ml volumetric flask and made up to volume with dd.H2O.
  • Tris-EDTA-NaCl buffer [TEN: 25 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH 7.6] 3.03 g Tris, 0.29 g EDTA and 8.77 g NaCl are dissolved in dd.H2O made up to 1 litre without pH adjustment and autoclaved.

Procedure for Protein transfer

  1. Following SDS-PAGE, the gel is dismantled from the electrophoresis unit, submerged briefly in ice cold transfer buffer; it is then laid flat on pre-wetted Hybond™-C nitrocellulose hybridisation transfer membrane (0.45 μm) supported on three layers of transfer buffer-wetted filter paper resting on the anode. The gel is overlaid with three wetted filter papers (or blotting paper), and then the cathode or another layer of nitrocellulose/gel/blotting paper. Care should be taken to exclude bubbles between gel and nitrocellulose, and between nitrocellulose and paper. The assembly is placed in a plastic tray resting on anode.
  2. The transfer cassette (Hoefer® TE series Transphor Electrophoresis Unit) is assembled; the nitrocellulose/gel/blotting paper assembly is placed in a plastic tray resting on anode. The ANODE (electrode on the nitrocellulose paper side of the assembly) is connected to anode connector and the gel side CATHODE to the cathode connector of an appropriate powerpack.
  3. The chamber is filled with ice-cold transfer buffer and stirred with a magnetic stirrer throughout the run.
  4. A current of 500 mA is passed for 20-30 min to effect transfer. Transfer is performed at a constant current of 30V over night, using Paul Meyers electroblotting apparatus.
  5. After transfer, the gel and the membrane are disassembled from the cassette and the outline of the gel is marked on the membrane.
  6. The membrane is rinsed with dd.H2O and then rinsed with Ponceau S to visualise the MR markers, the sample proteins and to assess the efficiency of the transfer. The positions of the lanes and the markers are marled by pricking the membrane gently with a needle point.
  7. The Ponceau S was decolourised with TEN and the membrane was air-dried before probing, or alternatively stored in a desiccator at 4°C.

NB: Do not touch assembly while power is on!!!

2. Immunoassay

A. Chromogenic blots

The attachment of specific antibodies to specific immobilised antigens on the membrane support can be visualised by indirect enzyme immunoassay techniques using chromogenic substrate which produces an insoluble product. Substrate products for chromogenic blots should also be light stable and easily visible after photography and/or electronic scanning. The enzymes used most frequently in western blotting are alkaline phosphatase and horseradish peroxidase (HRPO). Glucose oxidase and β-galatosidase have also found applications. Common alkaline phosphatase substrate is 5-Bromo-4-chloro-3-indolyl phosphate (BCIP). Cleavage of the phosphodiester bond produces an indoxyl that can undergo oxidation into an insoluble blue dimmer called 5,5’ dibromo 4,4’ dichloro indigo. The colourless tetrazolium salt, Nitro blue tetrazolium (NBT), is converted into an insoluble purple formazan upon reduction by the electrons liberated by the indoxyl.

The procedure below describes detection of proteins and alkaline phosphatase- conjugated secondary antibody, detected using 5-bromo-4-chloro-3-indolyl phosphatase (BCIP) and Nitro-blue tetrazolium (NBT) salts.

Reagents

  • Washing buffer: Tris-EDTA-NaCl buffer [TEN: 25 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH 7.6] 3.03g Tris, 0.29 g EDTA and 8.77 g NaCl are dissolved in dd.H2O made up to 1 litre without pH adjustment and autoclaved.
  • Blocking solution: [5% (w/v) non-fat milk powder in TEN]. 5 g non-fat milk powder is dissolved in 100 ml TEN.
  • Alkaline phosphatase substrate solution [0.015% (w/v) BCIP, 0.03% (w/v) NBT in detection buffer]. 1.5 mg BCIP in 1 ml dimethyl formamide (DMF) and 3 mg NBT are dissolved in 10 ml detection buffer. Prepared just before use.
  • Alkaline phosphatase detection buffer [50 mM Tris-HCl, 5 mM MgCl2 , pH 9.5). 0.61 g Tris and 0.10 g MgCl2.6H2O are dissolved in 90 ml dd.H2O. The pH is adjusted to 9.5 with HCL then the volume is made up to 100 ml with dd.H2O.
  • Endogenous phosphatase quenching solution. Levamisole (0.05 g) dissolved in blocking solution (100 ml) just before use.

Procedure

  1. Nitrocellulose blots are briefly rinsed in transfer buffer; then soaked for 1 hour at 37°C, or 2 hr at room temperature in blocking buffer containing 2 mM Levamisole. This allows the saturation of all non-specific protein binding sites on the blots.
  2. The membrane is washed 3*5 min in 10 ml 3% milk-TEN/wash.
  3. For attachment of antibody, the blots are incubated in primary antisera diluted 1/10-1/1000 in blocking solution [or 3% (w/v) milk-TEN solution], in sealed boxes for 2 hours on a shaking waterbath at room temperature (22°C).
  4. Blots are washed by shaking in 10 ml 3% milk-TEN/wash, 3*5 min, at room temperature. (Increase number and duration of washes if background is a problem).
  5. Probing of antibody binding; Blots are incubated in suitably diluted [3% (w/v) milk-TEN solution] alkaline phosphatase-conjugated secondary antibody for 1 hour.
  6. Membrane blots are then washed as before, immersed in the appropriate substrate solution [10 ml/100 cm2 blot] and developed until distinct bands are observed against a lightly coloured background.
  7. The membrane is then rinsed with dd.H2O, dried and kept in the dark until photography.

Table 1: A summary for probing and detecting blotted proteins by chromogenic detection system

Step Solution Volume Incubation time
1. Block and quench 3% milk-TEN + Levamisole 20 ml 1 hour
2. Wash 3% milk-TEN 3 * 10 ml 3 * 15 min
3. Primary antibody 3% milk-TEN 5 ml 2 hours
4. Wash 3% milk-TEN 3 * 10 ml 3 * 15 min
5. Secondary antibody 3% milk-TEN 10 ml 1 hour
6. wash TEN 3* 10 ml 3 * 15 min
7. Develop Substrate solution +10 ml + 1 hour
9. Wash (rinse) dd. H2O 10 ml 1 min
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B. Enhanced Chemiluminescence (ECL) blots

Chemiluminescence refers to the emission of blue light resulting from the (Horseradish peroxidase) HRPO/H2O2-mediated oxidation of cyclic diacylhydrazides (such as luminal). HRPO oxidises a peracid salt (in the presence of luminal) which leads to a raised oxidation state of the HPRO haem group. Upon returning to the ground state, luminal radical is formed and emits light as it decays. In ECL, an enhancer molecule (such as phenol) is included in the reaction mixture to react with the HRPO haem group in place of luminal. This leads to the formation of phenol radicals that in turn reacts to form luminal radicals and light is emitted in the manner as with the unenhanced chemiluminescence. ECL is faster and is highly stable (t1/2 ~60 min). The light emission from the HRPO-linked secondary antibody is detected using blue-light sensitive X-ray film (λmax = 428 nm). With this method, multiple exposures can be taken due to the slow decay rate of light. Since no insoluble substrate products are produced in ECL, the membranes can be stripped and reprobed a number of times following thorough washing to remove substrate.

The procedure below describes detection of proteins and alkaline phosphatase- conjugated secondary antibody, detected using Horseradish peroxidase ECL system.

Reagents

  • Tris-EDTA-NaCl buffer. [TEN: 25 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH 7.6]. 3.03 g Tris, 0.29 g EDTA and 8.77 g NaCl are dissolved in dd.H2O and made up to 1 litre without pH adjustment and autoclaved
  • 1 M MgCl2 stock solution. 50.83 g MgCl2.6H2O is dissolved in dd.H2O to a final volume of 250 ml and autoclaved.
  • 5 M NaCl stock solution. 73.05 g NaCl is dissolved in dd.H2O to a final volume of 250 ml and autoclaved.
  • Blocking solution. [3% (w/v) non-fat milk powder in TEN, pH 7.6]. 3 g non-fat milk powder is dissolved in 100 ml TEN just before use. [2% (w/v) BSA in TEN can also be used]
  • Peroxidase quenching solution. NaN3 (0.1 g) was added to blocking solution (100 ml)
  • ECL substrate solution. Equal volumes (500 μl) of detection solution 1 and 2 (containing H2O2 and luminol) were mixed before use.

ECL Procedure

The ECL procedure is essentially the same as for chromogenic blots, but table 2 below highlights some of the differences.

Table 2: Procedure for probing and detecting blotted proteins by ECL

Step Solution Volume Incubation time
1. Block and quench 3% milk-TEN + NaN3 20 ml 30 min
2. Wash 3% milk-TEN 3 * 10 ml 3 * 15 min
3. Primary antibody 3% milk-TEN 5 ml 2 hours
4. Wash 3% milk-TEN 3 * 10 ml 3 * 15 min
5. Secondary antibody 3% milk-TEN 10 ml 45 min
6. wash TEN 2 * 10 ml 2 * 15 min
7. Wash TEN + 200 mM MgCl2 2 * 12,5 ml 2 * 15 min
8. wash TEN + 1 M NaCl 10 ml 5 min
9. Wash dd. H2O 10 ml 1 min
10. Expose ECL solution 125 μl/cm 2 30 s – 10 min
Reference: UCT MCB protocols