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Western Blot General Protocols



The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.








I. Background

II. Direct Western Blot
III. Indirect Western Blot
IV. Troubleshooting Western Blot



I. Background

    Western Blot is a technique that uses antibodies for the specific recognition of a chosen antigen in a solution containing numerous proteins and other components, relying on the separation of the specific antigen from other substances based on molecular weight and the specificity of antibody-antigen recognition. Western Blot relies on the standard methods of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In general, SDS-PAGE is conducted in flat minigels, using either reducing or non-reducing buffers based on the presence or absence of disulfide bond breaking reagents such as beta-mercaptoethanol or dithiothreitol.


Direct Western Blot is applicable for primary antibodies already conjugated with a detection system. Direct Western Blot has advantages in that the method is rapid because only one antibody is used. Any cross-reactivity of the secondary antibody is also eliminated. The disadvantages of this method are 1) the affinity of the primary antibody may be reduced by conjugation and 2) there is little signal amplification. Indirect Western Blot is the more frequently used application. The advantages of this method are: 1) the reactivity of the primary antibody is preserved, 2) a wide variety of secondary antigen conjugations are available, and 3) sensitivity is increased by multiple binding of the secondary.

Indirect Western Blot requires a secondary antibody bearing the conjugate of choice for detection. The disadvantages of indirect Western Blot include the fact that cross-reactivity between the secondary antibody and other proteins can lead to high background signals, and the extra step takes additional time.


II. Direct Western Blot

A. Materials

1. Transfer Buffer: 25mM Tris base, 0.2M glycine, 20% methanol, pH 8.5.

2. SDS Sample Buffer (1X): 62.5mM Tris-HCl, pH 6.8 at RT, 2% SDS, 10% glycerol, 50mM DTT, 0.01% bromophenol blue or phenol red.

3. 10X TBS: To prepare 1 liter of 10X TBS: 24.2g Tris base, 80g sodium chloride. Adjust pH to 7.6 with HCl. Use at 1X.

4. Blocking Buffer: 1X TBS, 0.1% Tween-20, 5% nonfat dry milk. For 150ml, add 15ml 10X TBS to 135ml water, mix. Add 7.5g nonfat dry milk and mix well. While stirring, add 0.15ml Tween-20 (100%).

5. Wash Buffer TBS/T: 1X TBS, 0.1% Tween-20

B. Sample preparation and separation

1. Boil ~20ug sample in SDS sample buffer for 5 minutes and resolve on SDS-PAGE (12-15%).

2. Transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.

C. Blocking step

3. Block the membranes for 1 h at RT with 5% nonfat dry milk in Tris-buffered saline-Tween (TBS-T) for 20 minutes at RT with constant agitation.

4. Wash 3 times in TBS-T.

D. Primary antibody incubation

5. Incubate the membrane with the primary antibody (diluted in blocking buffer) for 2 hours at RT or overnight at 4°C.

6. Wash 3 times in TBS-T.

E. Color development

7. Detect using enhanced chemiluminescence detection method.


III. Indirect Western Blot

A. Materials

1. Transfer Buffer: 25mM Tris base, 0.2M glycine, 20% methanol, pH 8.5.

2. SDS Sample Buffer: 62.5mM Tris-HCl, pH 6.8 at RT, 2% SDS, 10% glycerol, 50mM DTT, 0.01% bromophenol blue or phenol red.

3. Blocking Buffer: 1X TBS, 0.1% Tween-20, 5% nonfat dry milk.

4. Wash Buffer TBS/T: 1X TBS, 0.1% Tween-20

5. For secondary antibody, select a product directed at the isotype of the primary antibody (anti-mouse IgG1, anti-rabbit IgM, anti-chicken IgY, etc.); conjugate as desired.

B. Sample preparation and separation

1. Boil ~20ug sample in SDS sample buffer for 5 minutes and resolve on SDS-PAGE (12-15%).

2. Transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water.

C. Blocking step

3. Block the membranes for 1 h at RT with 5% nonfat dry milk in Tris-buffered saline-Tween (TBS-T) for 1 hour at RT with constant agitation.

4. Wash 3 times in TBS-T.

D. Primary antibody incubation

5. Incubate membrane with primary antibody (diluted in blocking buffer) for 2 hours at RT or overnight at 4°C.

6. Wash membranes 3 times in TBS-T.

E. Secondary antibody incubation

7. Dilute secondary antibody in TBS-T with 1% nonfat dry milk and incubate for 1 h at RT.

8. Wash 3 times for 5 minutes each in TBS-T.

F. Color Development

9. Detect using enhanced chemiluminescence detection method.


IV. Western Blot Troubleshooting

A. No signal

1. The primary antibody and the secondary antibody are not compatible. Use secondary antibody that was raised against the host species of the primary antibody (e.g., If the primary is raised in mouse, then use anti-mouse IgG secondary antibody).

2. Insufficient antigen. Load at least 20-30ug protein per lane; Use protease inhibitors; Run the recommended positive control.

3. The primary antibody does not recognize the protein in the species being tested. Run the recommended positive control.

4. Too little primary or secondary antibody is bound to the antigen of interest. Use more concentrated antibody and/or incubate for longer time (e.g. overnight at 4ºC).

5. Excessive washing of the membrane. Do not over wash the membrane.

6. Too much blocking does not allow you to visualize your protein of interest. Instead of using 5% milk in the antibody buffers try removing the milk or using 1%. Switch blocking reagents or block for less time.

7. Secondary antibody inhibited by sodium azide. Do not use sodium azide in conjunction with HRP-conjugated antibodies.

B. High background

1. Insufficient blocking. Increase the blocking incubation period and consider changing blocking agent. Usual recommendations are for 5% non-fat dry milk, 3% BSA, or normal serum for 30 min. These can be included in the antibody buffers as well.

2. Primary antibody concentration is too high. Examine multiple dilutions of primary antibody for optimum performance. Incubate for longer times, but in more dilute solution.

3. The secondary antibody may be binding non-specifically or reacting with the blocking reagent. Run a secondary control without primary antibody.

4. Cross-reaction between blocking agent and primary or secondary. For phosphoproteins, use BSA as a blocking reagent instead of milk.

5. Washing of unbound antibodies may be insufficient Increase the number of washes.

C. Multiple bands

1. The protein sample may have multiple modified forms in vivo such as acetylation, methylation, myristylation, phosphorylation, glycosylation etc. Examine the literature and use an agent to dephosphorylate, de-glycosylate, etc. the protein to bring it to the correct size.

2. The target in your protein sample has been digested (if observe bands at lower molecular weight than expected). Make sure that you incorporate sufficient protease inhibitors in your sample buffer.

3. The protein target may form multimers (if observe bands at higher molecular weight than expected. Try boiling in SDS-PAGE for 10 minutes rather than 5 minutes to disrupt multimers.

4. Primary antibody concentration is too high. Multiple bands are often seen at high concentrations. Decrease the antibody concentration and/or the incubation period.

5. Secondary antibody concentration is too high. Secondary antibodies will bind nonspecifically at high concentrations. Decrease the concentration. Run a secondary antibody control (without the primary).

6. The bands may be non-specific. Use blocking peptides, when possible, to differentiate between specific and non-specific bands. Only specific bands should disappear when the blocking peptide is used.

D. White bands on a black blot

1. Concentration of primary and/or secondary antibody is too high. Dilute the antibodies further.