Possible Causes

What You Can Do?

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Protein post-translationally modified or alternatively spliced

Check if the protein of interest is post-translationally modified or alternatively spliced and produce other isoforms.

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Incomplete protein denaturation

Freshly add DTT or β-Mercaptoethanol to sample buffer, or adequaltely boil samples to ensure complete bond breakage between peptides.

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Membrane protein issue

If a membrane protein is to be detected, try low temperature (~65°C) or avoid boiling which might cause aggregation of membrane proteins.

Possible Causes

What You Can Do?

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Protein post-translationally modified or alternatively spliced

Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and store in moist chamber at 4°C. 

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Protein degradation

Keep the voltage and temperature low while running SDS-PAGE. If necessary, run gel in the cold room. Remove bubbles trapped at the bottom of gel to ensure even electrophoresis.

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Protein Multimerization

Make sure that the salt concentration of lysis buffer is kept between 0.15M to 0.5M.

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Antibody concentration too high

Make sure that total amount of protein loaded into each well is between 20-50μg.

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Antibody issue

Optimize the dilution factor of primary and secondary antibody.

While performing Immunoprecippitaiotn (IP) experiment, make sure that the secondary antibody used to detect the protein of interest is derived from a species different from that of antibody used to pull down protein. Alternatively, use a secondary antibody that recognize only the native form of IgG to detect IP protein.

Possible Causes

What You Can Do?

►

Reagent contaminated

Prepare all reagents freshly.

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Blocking agent insufficiently dissolved

Make sure that the blocking agent such as milk or BSA is completely dissolved before use. Alternatively, filter the blocking solution with 0.45μm filter before use.

Possible Causes

What You Can Do?

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Poor gel preparation

Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and store in moist chamber at 4°C. 

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Voltage, temperature too high, field effect

Keep the voltage and temperature low while running SDS-PAGE. If necessary, run gel in the cold room. Remove bubbles trapped at the bottom of gel to ensure even electrophoresis.

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High salt concentration in samples

Make sure that the salt concentration of lysis buffer is kept between 0.15M to 0.5M.

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Protein overloaded

Make sure that total amount of protein loaded into each well is between 20-50μg.

►

Antibody concentration too high

Optimize the dilution factor of primary and secondary antibody.

Possible Causes

What You Can Do?

►

Poor gel preparation

Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and stored in moist chamber at 4°C. 

►

Protein overloaded

Make sure that total amount of protein loaded into each well is between 20-50μg.

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High membrane protein concentration in samples

If membrane fraction is used, make sure that sample is sufficiently diluted before loading into SDS-PAGE.

Possible Causes

What You Can Do?

►

Antibody concentration too high

Optimize the dilution factor of primary and secondary antibody.

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Insufficient washing

Make sure that the blot is washed in sufficent washing buffer. Increase washing time or the percentage of Tween-20 if necessary.

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Insufficient blocking

Make sure that the blot is sufficiently blocked. Increase the percentage of skimmed milk up to 5% if necessary.

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Improper blocking buffer used

For the detection of phospho-proteins, use BSA instead of milk as blocking agent.

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Membrane dried out

Make sure that the membrane is moist throughout the whole process of western blot.

Possible Causes

What You Can Do?

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Protein not expressed in the sample used

Make sure that the protien of interest in sufficiently induced in the sample used. Fractionation might be necessary for some proteins expressed in particular organelles.

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Inadequate/incomplete transfer

Be sure that the transfer is adequate, especially for high molecular weight proteins. Make sure that the transfer is complete by staining the membrane with Ponceau S solution or using a pre-stained marker as indicator.

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Antibody issue

Make sure that primary antibody dilution and the incubation condition is optimal. Compatible secondary antibody should be used.

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Poor activity of ECL

Prepare ECL solution freshly prior to detection.

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Sodium Azide interference

Make sure that there is no Sodium Azide in the antibody dilution buffer. Wash the blot thoroughly before adding ECL.