+44 (0) 1223 755950
+1 832 327 7413
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). |