| Problem |
Cause |
Solution |
| 1. High background |
a. Non-specific binding to agarose beads/antibodies./td> |
a. Add a pre-clearing stage to prevent non-specific binding. |
|
b. Protein aggregates that are not detergent soluble. |
b. Extract the supernatant immediately after centrifugations. |
|
c. Too much antibody used can leads to non-specific binding. |
c. Check the recommended amount of antibody.. |
|
d. Inadequate washing. |
d. Wash well when relevant and use more stringent washes. As well as increasing the amount of washes. |
|
e. Non-specific proteins are binding with the beads. |
e. Make sure beads are pre-blocked enough in 1% BSA in PBS. |
|
e. Frozen cells were used. |
e. Try to use fresh material but if not use frozen lysates as opposed to frozen cells. |
| 2. Specific antigen is not being detected |
a. The antibody used is not suitable. |
a. Try using a different antibody for example polyclonal antibodies may work better than monoclonal ones. |
|
b. Inappropriate beads used so do not bind antibodies. |
b. Check the type of beads that should be used for the antibody isotype. |
|
c. Washes are too harsh meaning antibodies were stripped from agarose beads. |
c. Reduce detergent and salt, alternatively use a different detergent and reduce the number of washes. |
|
d. Antigen of interest is not present or there is a low level. |
d. Ensure the sample is appropriate and, if there is a low level, increase the amount of lysate used. |
|
e. Too many proteins competing in the mixture. |
e. Before adding the antibody make sure to centrifuge to remove insoluble proteins. |
|
f. Antibody concentration too low. |
f. Titrate antibodies to find appropriate amount required. |
|
g. Incubation times not sufficient. |
g. Increase incubation times (overnight at 4oC). |
