I have purified my protein (Metallo beta Lactamase, ~28 kDa) by Ni-NTA column. After running it in 15% SDS gel (maintaining standard protocol), I am not getting any band. What could be the possible reason?
1. Gel problems.. did the ladder look ok? Didnt get hot or anything?
2. Proteases ...
3. Disulfide bonds
4. Crud on platinum electrodes when running gel
5. Running Buffer off
6. Electrodes backwards
Lots of things I guess...
I thought of another .. try to load more protein,...
Use silver stain, very sensitive.
sorry I could not be of more help
In my opinion, the SDS gel conc ist ok (12% would also be sufficient).
The protocol (of the Ni-NTA procedure as well as the WB) should be revised in detail. Maybe you have protein loss during purification (via Ni-NTA, wrong washing or eluting step/buffer etc.) or during WB (transfer to the membrane (semi-dry of Tank blot), choice ob membrane, voltage; or due to other mistakes (wrong washing and/or transfer buffer, to long fractionation time/period etc.).
Andreas Eisenreich Regarding the concentration of protein, I can tell you it is high enough, 96μM. Another thing is- the protein is showing a prompt response to the assay with nitrocefin (as this is a beta lactamase). So even if some amount has been lost, it is not a problem I think. Do you agree?
Subhecchha,
I agree, with some small loss. You see good response with assay, so its there....
Can you try to do a Silver-Stain.
I have done this, and it works well, vs a comassie blue stain.
https://www.researchgate.net/post/good_protocol_for_silver_staining
Article Silver staining of proteins in Polyacrylamide gels
The mentioned results suggest this. However, I think that there is a (at least one) problem regarding the method. Maybe, loss during transfer of proteins to the membrane (have you done any control experiments (transfer of other protein samples not got by Ni-NTA method), transfer of stained protein marker/ladder, Ponceau staining of gel and mebrane after transfer?). Have you checked whether there are possible sources of uncertainty in the protocol (vs standard protocols or experiments done by others)?
Andreas Eisenreich , I haven't done any control experiment regarding this. First method we use to purify protein in our lab is Ni-NTA, the we go for SEC.
There is many probabilities:
Increase the amount of the protein
Decrease the concentration of the gel
Decrease the voltage to 90 or 100
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