Possible Cause & Solution
a. Non-specific antibody binding.
Reduce primary antibody concentration
Decrease the amount of total protein loaded on gel
Adjust membrane blocking conditions
Increase number of washes
Verify the specificity of the antibody
Blot with the secondary antibody alone. If bands develop, choose an alternate secondary antibody
b. Degradation of protein.
Prepare fresh samples. Use protease inhibitors during sample preparation. Minimize freeze/thaw cycles of sample.
c. Aggregation of analyte.
Increase the amount of DTT (20 -100mM) to ensure complete reduction of disulfide bonds. Heat in boiling water bath for 5-10 minutes before loading onto gel.
Cell lines that have been passaged many times may accumulate differences in their protein expression profiles. Go back to the original non-passaged cell line and run the current and original cell line samples side-by-side.
The protein sample has multiple modifications in vivo such as acetylation, methylation, glycosylation, phosphorylation, etc. Review the literature for modified protein variants. Adjust sample preparation accordingly.
Target protein has multiple isoforms, or other proteins share similar epitopes. Check the literature for target protein isoforms. Perform a BLAST search to check for possible cross-reactions. Include other cell or tissue types.