Possible Cause & solution
a.The cell or tissue type does not express the protein of interest. Perform a positive control, preferably from cell or tissue lysate already verified to express the target protein.
b. Improper cell treatment.
Stimulate cells with the appropriate chemical, protein, etc. Verify that the stimulation works.
c. Improper sample preparation for gel loading.
Ensure that the protein in the lysate is stable. Use appropriate protease and phosphatase inhibitors, etc. Ensure protein samples contain SDS, and have been heated prior to gel loading. Include a reducing agent such as dithiothreitol (DTT) and/or 2-mercaptoethanol.
d. Insufficient amount of antigen present.
Load more protein on gel. Also, if the specific antigen concentration is too low, try enriching the antigen by fractionation or by immunoprecipitation.
e. Proteins did not transfer properly to the membrane.
Wet PVDF membrane in methanol or nitrocellulose membrane in transfer buffer before use
Ensure there is good contact between the membrane and the gel
Optimize the transfer time
After transfer, ensure molecular weight markers were transferred; stain the membrane with Ponceau red, and the gel with Coomassie blue
f. Insufficient antigen binding to membrane
If the antigen has low molecular mass, it may pass through the membrane. Switch to a membrane with a smaller pore size, or switch to a different type of membrane.
g. The antigen is masked by the blocking buffer
Test different blocking buffers, such as milk, serum, BSA in Tris-buffered saline and phosphate-buffered saline. Test different concentrations of each.
h. Insufficient amount of antibodies present
Increase concentration of primary and/or secondary antibody.
i. Antibody exposure time is too short.
Increase the exposure time.
j. Antibodies may have lost activity.
Test antibodies by performing a Dot Blot.
k. Excessive washing of the membrane.
Reduce the number of washes.
l. Substrate incubation is too short.
Increase substrate incubation time.
m. Substrate has lost activity
Test substrate using a positive control.
n. Sodium azide is inhibiting enzyme reaction of HRP-conjugated antibody.
Do not use sodium azide together with HRP-conjugated antibodies.