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Troubleshooting Western Blots

  The Molecular Station Guide to Western Blotting Problems and Solutions: Common Western Blot Problems and Solutions for them!

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What is your western blot problem? - Organized Alphabetically

Fuzzy Bands

Low Signal

Membrane is Glowing in the Dark Room

High Background

Spots on Film

Too many Bands in Western Blot

Weak Signal

 

 

Troubleshooting Western Blots Problem:

Spots on Film (missing bands) :

- Poor Transfer due to air bubbles during transfer to membrane

- dirty film when adding to the developer

- developer rolls are dirty

Solution to spots on film:

- use a pasteur pipette as a roller. roll the membrane and gel before transfer to remove bubbles. keep membrane and materials wet.

- keep film clean before adding to the developer

 

Troubleshooting Western Blots Problem:

Too Many Bands in Western Blot :

- usually due to many non-specific bands

- poor primary antibody quality or old antibody

- not enough blocking

- proteolytic cleavage of antigen - all additional bands are lower MW than your protein, ( ie your protein is being detected but was degraded)

Solutions to too many bands on western blot:

- purchase another antibody or replace with a fresh antibody stock

- block with 5% dry non-fat milk or BSA prior to adding primary. If you are already blocking, consider blocking during primary and secondary antibody incubations and also increasing blocking milk or BSA concentration.

- keep everything on ice, use fresh samples, store in - 80C, and use protease inhibitors such as PMSF

 

Troubleshooting Western Blots Problem:

High background; low signal to noise ratio on Western Blot

- black or every dark film; film was exposed too long

- blocking was insufficient ; non-specific binding of primary antibody and secondary antibodies were not washed completely

- washing was insufficient

- film glows in dark! - too high secondary or primary dilutions

Solutions to Dark Film:

- expose for less time, decrease exposure time

- increase blocking duration of non-fat dry milk 5% incubation or increase the concentration.

- also you can try blocking with whole serum of the host animal with (or before) the secondary antibody

- increase washing times and volumes to help remove any non-specific signal due to weak antibody binding.

- using a stronger detergent to wash - Instead of TBST (Tween-20), use stronger detergents such as NP-40 or SDS which may provide a more stringent wash and reduce background (do this only if you band is strong! - washing also removes some your band of interest!)

- very high secondary antibody (or even primary) concentration - perform optimization experiments to determine proper antibody dilutions. dilute the antibodies further.

 

Troubleshooting Western Blots Problem:

Low Signal or Weak Signal

- due to not enough protein loaded on the gel

- primary antibody is old or not good

- phospho-proteins usually need overnight primary antibody incubation

- not enough secondary antibody

- over-blocking

- developing reagents are bad / you made a mistake when preparing them

Solutions to Low Signal or Weak Signal:

- load more protein sample onto gel

- try fresh primary antibody

- incubate primary antibody overnite for phosphoproteins at 4 degrees C (cold room shaker)

- increase concentration of protein (lyse samples in less lysis buffer)

- decrease concentration of blocking agent

- check the developing reagents; prepare fresh ECL and re-ECL the membrane

- increase the concentration or the length of primary antibody incubation period

- increase concentration or the length of secondary antibody incubation period

 

Fuzzy Bands, Bands Smeared, Bands not Sharp

- bands smeared due to hot gel

- bands fuzzy due to high voltage

Solutions to Fuzzy bands and Un-sharp Bands:

- decrease voltage or current, run in cold room, and prepare new running buffer. ( heat causes the gel to lose its rigidity and its resolving power)

- pre-soak transfer membrane in the appropriate transfer solution (determined by the membrane manufacturer) for the required length of time.