| Possible Cause |
Solution |
| 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. |
| Improper cell treatment. |
Stimulate cells with the appropriate chemical, protein, etc. Verify that the stimulation works. |
| 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. |
| 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. |
| 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
|
| 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. |
| 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. |
| Insufficient amount of antibodies present |
Increase concentration of primary and/or secondary antibody. |
| Antibody exposure time is too short. |
Increase the exposure time. |
| Antibodies may have lost activity. |
Test antibodies by performing a Dot Blot. |
| Excessive washing of the membrane. |
Reduce the number of washes. |
| Substrate incubation is too short. |
Increase substrate incubation time. |
| Substrate has lost activity |
Test substrate using a positive control. |
| Sodium azide is inhibiting enzyme reaction of HRP-conjugated antibody. |
Do not use sodium azide together with HRP-conjugated antibodies. |
| Possible Cause |
Solution |
| 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
|
| Degradation of protein. |
Prepare fresh samples. Use protease inhibitors during sample preparation. Minimize freeze/thaw cycles of sample. |
| 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. |
| Possible Cause |
Solution |
| Too much protein per lane. |
Titrate down the amount of protein loaded per lane. |
| Insufficient blocking of non-specific binding. |
Adjust blocking conditions. Include blocking agent in the antibody buffers as well. |
| The primary antibody concentration may be too high |
Titrate the antibody to find the optimal concentration. |
| The secondary antibody may be binding non-specifically. |
Blot with the secondary antibody alone. If bands develop, choose an alternate secondary antibody. |
| Incubation temperature may be too high. |
Incubate blot at 4°C. |
| Cross-reaction between blocking agent and primary or secondary antibody. |
Add a mild detergent, e.g. Tween® 20, to the incubation and washing buffers for phosphoprotein specific antibodies. Use BSA as a blocking reagent instead of milk. Milk contains casein, a phosphoprotein, and your antibody may be cross-reacting with the casein. |
| Washing of unbound antibodies may be insufficient. |
Increase the number of washes. |
| The membrane may give high background. |
Nitrocellulose membrane may give less background than PVDF. |
| The membrane has dried out. |
Avoid drying out the membrane during processing and incubation. |
