There's is a very thick band on the ladder (NOT on the sample lane!) when I tried for p21 western. Is this because of p21 primary antibody (H164).? Is there any measures to get good blots..!
Mh, dont forget: your marker consists of various size defined proteins which are coupled to whatever dyes. Either it is a crossreaction with your 1st antibody or with the 2nd ab. Easy to test though. In our marker one of the proteins seems to be out of rabbit - If we incubate our membran with 2nd anti-rabbit ab, 35 kD band always comes up. :)
Try to use a different ladder. Problem should be gone then. If you dont have the possibility, leave one lane between ladder and samples free.
Good luck... You know, there are the good, the bad and the ugly Westerns :)
I am learning to do a good western myself. According to my experience, loading 10 ul of marker gives a perfect marker lane. Blocking for 2 hours, primary antibody- overnight, secondary antibody 2-3 hours are the basic rules which everybody knows. Vigorous washing multiple times after primary and secondary antibody incubation, not letting the membrane to dry, applying minimal detecting agent, are few things i have learned to get a satisfactory blots.
And as Teresa said, you can fill a loading buffer between the marker and your sample. There is a possibility of sample spillage in the marker channel while loading sample.
There are lot of trouble shooting guides you can go through.
Happy western blotting!!!
Check the quality of the transfer membrane and the filter pads used. If there is any dirt or if you have reused the filter pads, there is a possibility that some artifacts are possible. Next use less amount of the ladder and take care of the sample spillage. Hopefully this helps
- Badges
- Science method