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Western Blot Protocols (part 3) - Antibody Incubation & Gel Visualization

Learn more about western blot, please see Previous Section.

The antibody incubation process of western blot after protein transfer onto membrane

Figure 1. The antibody incubation process of western blot after protein transfer onto membrane.


Visualization of proteins in membranes (optional):
As an optional step, we can verify the protein were transferred successfully by staining the membrane with ponceau red. Incubate the membrane in ponceau for 5 minutes and wash with water until the bands are clear. After verification, the bands can then be destained by continuing to wash with water or TBS-tween until the dye is completely removed. When using a PVDF membrane, re-activate the membrane with methanol then wash again in TBS-Tween. (Figure 1 1a, 1b, 1c)
As non-specific binding of antibodies to the membrane is detrimental to the specificity and sensitivity of the assay, it is essential to "block" spaces not already occupied by proteins. Choice of blocking strategy will be guided by samples and the antibodies used. The most common permanent blocking agents include bovine serum albumin (BSA), non-fat milk, normal goat serum, casein and fish gelatin (Table 1.).

Table 1. Proteins used as blocking agents in Western blotting

Protein Recommended concentration Buffers Membrane compatibility
BSA 0.2-5% (W/V) Tris-buffered saline (TBS)/phosphate buffered saline (PBS) Nitrocellulose Polyvinylidine difluoride (PVDF)
Non-fat milk 3-5% (W/V) TBS, PBS Nitrocellulose
Amersham ECL Prime Blocking Agent 2-5% (W/V) TBS, PBS Nitrocellulose
Casein 1% (W/V) TBS Nitrocellulose
Fish gelatin 2-10% (W/V) TBS, PBS Nitrocellulose
Serum 1-5% (V/V) TBS, PBS Nitrocellulose

As each antibody-antigen pair has unique characteristics, no single blocking agent is ideal for every Western blotting process. Determining the best blocking agent and optimal concentration are key steps for the success of immune detection. PBS or TBS are commonly used as buffers for blocking agents. Common blocking buffers including 5% non-fat dry milk or BSA in a TBS-tween solution. However, do not use dry milk solution when probing with phosphor-specific antibodies, as it can cause high background from its endogenous phosphoprotein, casein. It is important to soak the blotted membrane in freshly prepared blocking agent for 30 min to 2 h at room temperature with constant agitation. Alternatively, soaking the membrane for 1 h a 37°C or overnight at 4°C can help solve some persistent background issues. Decant the block solution and wash with TBS-tween for 5 minutes.

2.Primary antibody incubation

Following the blocking step, the protein of interest can be detected using antibodies. This following process are very similar to ELISA. Many tips and cautions in ELISA are equally applied in western blot. Both monoclonal and polyclonal antibodies can be used for Western blotting analysis (Table 2). The most two important standard while choosing an antibody are: 1) weather it can recognize the denatured proteins; and 2) weather it can cause cross reaction. Polyclonal antibodies tend to be more sensitive, but are less specific than monoclonal antibodies. Monoclonal antibodies, on the other hand, tend to be more specific but less sensitive. Polyclonal antibodies are usually chosen for their relatively lower price and less time consuming to produce.

Primary antibodies should be raised in species as distinct as possible from the sample species: it is better to raise a primary antibody against a mouse protein in a rabbit, for example, rather than a rat.

Table 2. Difference between poly and mono clonal antibodies.

Signal specificity Advantages Disadvantages
polyclonal antibody good good, but have some background Most can recognize denatured protein Not easy to repeat, sometime with high background
monoclonal antibody vary between antibodies Best, but may have crossreaction Fine specificity, not limit by resource Most cannot recognize denatured protein
mixed monoclonal antibody Best Best Strong signal, fine specificity, not limit by resouce Easy to obtain

Dilute the primary antibody in a blocking buffer at the concentration recommended to the datasheet. Incubate overnight at 4 degrees Celsius with gentle shaking. A recommended option step is to also use a positive loading control antibody which allows the user to verify equal amounts of total protein were loaded into each well and aides in troubleshooting by removing any uncertainties with the western blot procedure. The next day, decant off the primary antibody solution and wash the membrane with large volumes of TBS-tween and vigorous agitation 5 times for 5 minutes each. These stringent washes are extremely important for removing non-specific background signals.
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3.Secondary antibody incubation

A wide variety of secondary antibodies are commercially available. The choice of secondary antibodies depend firstly on the species in which the primary antibody was produced. For example, the primary antibody was of the IgG isotype and produced in goat, the secondary antibody must be an anti-goat IgG antibody produced in another species as it will bind to the Fc region of the primary antibody. Although there is no strict rule, secondary antibodies raised in certain host species may lead to high background levels.
The procedures for incubation of the secondary antibody solution and the membrane are essentially similar to those described for the primary antibody. Dilute the secondary antibody in blocking buffer and incubate the membrane for 1h in room temperature at the concentration recommended on the data sheet. Decant secondary antibody and wash the membrane with large volume of TBS-Tween and vigorous agitation 5 times for 5 minutes each, and ready to the next detection phase.
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A variety of detection systems, based on chemiluminescence, chemifluorescence, fluorescence, chromogenic or radioisotopic detection are available. The coloration/visualization system are also very similar with ELISA. See Immunoassay and Chemiluminescence Immunoassay Guide for further understand the detection system.
The most common, most sensitive and most inexpensive detection method is the electrochemiluminescence (ECL) system. This method utilize the HRP enzyme, which was conjugated to the secondary antibody to catalyze the ECL reaction and produce light. The light is then gathered by detection machine and print onto x-ray film and developed or digitized with the aid of specialized CCD camera sensitive enough for detection. There are two kinds of ECL reagents: reagent A and reagent B, we mixed these two ECL reagents in 1:1 ratio, and incubate the membrane into the reagent for 3-5 minutes without agitation. After incubation, decant the ECL mixture and use a wipe to wipe off excess solution from the corner of the membrane. Place the membrane in a clear plastic wrap such as a sheet protector to prevent drying. Both film and camera system allow to manually adjust the exposure time in order to ensure a picture perfect western blot. Relative band density can be quantified with commercially available software. Proper molecular weight can also be verified by comparing band size to the molecular weight ladder.

Table 3. Commonly used Western Blot Reagents Recipe

30% Polyacrylamide
Acrylamide monomer:29g;
Methylene Diacrylamide:1g;
Dilute with ddH2O to 100ml volume at 37°C
1.5M Tris-HCl (PH8.8) Tris: 90.85g
Dissolve in 400 ml ddH2 and add to final volume of 500ml.
Use concentrated HCl to adjust pH to 8.8
1.0M Tris-HCl (PH6.8) Tris: 60.5g
Dissolve in 400 ml ddH2 and add to final volume of 500ml.
Use concentrated HCl to adjust pH to 6.8
0.5M Tris-HCl (PH6.8) Tris: 30g
Dissolve in 400 ml ddH2 and add to final volume of 500ml.
Use concentrated HCl to adjust pH to 6.8
10% SDS (PH7.2) SDS: 10.0 g, dissolve in 80ml ddH2O, and add to final volume of 100ml. keep 68°C to help dissolve.
Use concentrated HCl to adjust pH to 7.2
10% APS
(ammonium persulfate)
AP: 1.0g, dissolve in ddH2O and add to volume of 10ml.
Attention: AP can just keep in 2 weeks at 4°C, better use 0.2ml aliquots at -20°C for storage.
10×Electrophoresis Buffer Tris: 30.3 g;
Glycine: 144 g;
SDS: 10 g;
Dissolve in 800ml ddH2O and add to volume of 1L.
10×Transfer buffer
(without methanol)
Tris: 30.3 g;
Glycine: 144.1 g;
Dissolve in 900ml ddH2O and add to volume of 1000ml。
(Add less than 0.5% SDS for large protein)
1×Transfer buffer 1L (1×Transfer buffer)=100ml (10×Transfer buffer) + 700ml (ddH2O) + 200ml Methanol (Add before use)
10×TBS Tris: 24.23 g;
NaCl: 80.06 g;
Dissolve in 800ml ddH2O and add to volume of 1L.
Use concentrated HCl to adjust pH to 7.6
1×TBS-Tween 100ml 10×TBS, add 5ml 20% Tween 20 or 1ml Tween 20,
Add ddH2O to volume of 1L
(Attention: Add Tween 20 slowly along the beaker wall, or it will bring out bubbles.)
5% Blocking buffer
(non-fat milk)
Non-fat milk: 5g
Dissolve in 100ml TBST and well mixed.
Coomassie Brillant Blue solution (1L) Coomassie R-250: 1.0g
Methanol: 500ml
Glacial acetic acid: 100ml
ddH2O: 400ml
Could be store for 6 months at room temperature. Use filter paper to make filtration if precipitation.
10× Ponceau Red Ponceau red: 2g
Trichloroacetic acid: 30g
Sulfosalicylic acid: 30g
Dissolve in 100ml ddH2O
Destaining solution Methanol: 50ml
Glacial acetic acid: 70ml
Dissolve in 880ml dd H2O. store for 1 month at room temperature
2×SDS loading buffer
0.5M Tris-HCl: 2mL;
10% SDS: 4mL;
Glycerin: 2mL;
β-Mercaptoethanol: 140μl;
Bromophenol blue: 0.1mg
20% Tween 20
Stock solution
Add 20mL Tween 20 into 100mL 1×TBS

Western Blot Trouble Shooting
For the trouble shooting of common causes of unusual or unexpected bands, no bands, faint bands or weak signal, high background on the blot and more, please read our WB TROUBLESHOOTING TIPS


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