Western blot experimental experience summary

WB (western blot), which has been doing for a long time, has gone a lot of detours. In fact, it is not difficult to find out that WB wants to do well. It summarizes the problems that may be encountered in WB experiments, analyzes the possible causes and corresponding solutions. Can be the cornerstone of your experimental success.

Question 1: Insufficient film transfer

(1) The film is not completely evenly soaked

A 100% methanol permeable membrane was used.

(2) The molecular weight of the target protein is less than 10 000

Select a membrane with a small pore size to shorten the transfer time.

(3) The isoelectric point of the target protein is equal to or close to the pH of the transfer buffer.

Try using other buffers such as CAPS buffer (pH 10.5) or using low pH buffers such as acetate buffer.

(4) The methanol concentration is too high

Excessive methanol concentration causes the protein to separate from the SDS, thereby precipitating in the gel, while causing the gel to shrink or harden, thereby inhibiting the transfer of high molecular weight proteins. Reduce the methanol concentration or use ethanol or isopropanol instead.

(5) Insufficient transfer time

Extended transfer times are required for thick gels as well as high molecular weight proteins.

Question 2: High background

(1) The film is not completely evenly soaked

A 100% methanol permeable membrane was used.

(2) Insufficient film washing

Increase lotion volume and number of washes.

(3) Insufficient blockage

Increase the incubation time of the blocking solution or increase the temperature. Choose the appropriate blocking reagent (skimmed milk powder, BSA, casein, etc.).

(3) The concentration of the secondary antibody is too high

Reduce the concentration of the secondary antibody.

(4) Membrane drying during the test

Ensure sufficient reaction solution to avoid dry film.

(5) Overexposure

Reduce exposure time.

(6) The antibody cross-reacts with the blocking protein

The cross-reactivity of the antibody with the blocking protein is detected, and a blocking agent without cross-reaction is selected. The addition of Tween-20 to the wash solution reduces cross-reactivity.

Question 3: No positive bands

(1) Insufficient antibody staining

Increase antibody concentration and prolong incubation time.

(2) Enzyme inactivation

The enzyme and the substrate are directly mixed, and if no color is developed, the enzyme is inactivated. Select an active enzyme conjugate that is active during the period of validity.

(3) The target protein or target protein content in the specimen is too low.

A positive control is set. If the positive control has a result, but the specimen does not, the sample may contain no target protein or the target protein content is too low. Consider increasing the amount of sample loaded to address the low target protein content.

(4) Reagent mismatch

The primary antibody does not match the tissue species, the primary antibody and the secondary antibody or/and the substrate and the enzyme system. The effectiveness of the secondary detection system can be verified by setting an internal reference.

(5) Primary antibody failure

Select the antibody within the validity period; and select the current working solution.

(6) HRP inhibitors

Avoid using sodium azide in the solution and container used.

Question 4: There is a positive band, but the band is weak

(1) Insufficient antibody staining

Increase antibody concentration and prolong incubation time.

(2) Reduced enzyme activity

The enzyme and the substrate are directly mixed, and if no color is developed, the enzyme is inactivated. Select an active enzyme conjugate that is active during the period of validity.

(3) The target protein content in the specimen is too low

Increase the amount of specimen loaded.

(4) excessive film washing

Shorten the washing time.

(5) HRP inhibitors

Avoid using sodium azide in the solution and container used.

(6) Reduced antibody activity

Select the antibody within the validity period, and the working fluid is now ready for use, avoiding long-term placement.

(7) Over-closed

Reduce the amount of sealer or shorten the time; switch to different sealer types.

(8) The exposure time is too short

Increase the exposure time.

(9) HRP inhibitors

Avoid using sodium azide in the solution and container used.

Question 5: The strip position (size) is incorrect; or there is a non-specific strip

(1) Non-specific binding of secondary antibodies

Add a control: no additional antibody, other operations are unchanged, you can verify whether the background is from the source of the secondary antibody system. Choose other secondary antibodies (more specific, only for heavy chains).

(2) The specificity of the primary antibody is not enough

Monoclonal or affinity purified antibodies are used to ensure antibody specificity.

(3) Protein degradation

Freshly prepared specimens were used and protease inhibitors were used.

(4) Dimer or multimer exists

Increase protein denaturation process and strength.

(5) The antibody concentration is too high

Decreasing the concentration of antibodies (primary antibodies, secondary antibodies) can reduce non-specific bands.

(6) The protein loading is too large

Reduce the amount of sample loading.

Question 6: The background is spotted

(1) There are aggregates in the blocking agent

The blocking reagent is filtered prior to use.

(2) There are aggregates in HRP coupled secondary antibodies

The secondary antibody is filtered to remove aggregates.

Question 7: An anti-image appears on the film (white strip on a dark background )

(1) HRP content is too high

Reduce the concentration of the enzyme-linked secondary antibody.

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