My protein of interest is DAGLa (115 kDa). We received the antibody for this from another researcher who reliably shows bands at this weight. We do see a light banding at this weight, but the antibody picked up the majority at ~70 kDa and ~45 kDa. Why are we seeing the ladder fluoresce at ~70 kDa for us if it reliably picks up at 115 kDA?
Is there a way to test if the protein was cleaved?
Also, if I use a blocker against my protein of interest and there are no bands, can I assume the banding is to the correct protein, even though it is of low weight?
Hello,
If the antibody was previously shown to react the the protein of the expected size, as you mentioned, the best explanation is that under your experimental conditions, your target protein is degraded. What is the origin of your protein sample ? A native protein extract, a recombinant protein extact ? Did you use protease inhibitors before extraction ? Do you have a pure protein fraction available to test this ? Another possibility, is that the antibody aliquot at your disposal is damaged, and the main protein bands detected at lower MW are artifacts. Do you have preimmune serum to test this ?
Thank you for the reply!
We are using rat brain punches as our sample, and yes we used protease inhibitors in the extraction buffer. We are getting a blocker protein to run alongside as our next test, though if it shows no bands I am confused on how to proceed.
It could be cleaved or it is a matter of splicing. The best way to see what is going on is to cut out the bands and do protein sequencing or MS.
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