Usually for MS analysis we send bands from stained gels. Is there another method for identification of specific proteins? I wonder if it's possible to cut a piece of membrane with identified protein after WB and check it on MS?
Dear Hila,
it is possible to identify protein from WB but you need a MS-MS capable instrument because the sample is too complex to perform a simple Peptide MS fingerprint (PMF).
Some tips:
- The main problem with WB is that you cannot directly see the bands/spots of protein, if you use a fluorescent chemistry to highlight your Ab. So you must create a mask to cut in the right place or using a robot with camera for fluorescent dye.
- Use PVDF membrane because the nitrocellulose membrane dissolves in the solvents.
- The other problem usually is that you cannot perform buffer exchange since the proteins are loosely linked to the membrane (e.g. for reduction and alkylation).
- Last but not least, is better to use BSA and not milk to block the membrane, otherwise you get the milk proteomics.
Regards,
francesco
Dear Hila,
There are protocols developed to carry out generation and efficient extraction peptides from Nitrocellulose or PVDF membranes.
The key to success is to block the membranes before the addition of the protease; otherwise, trypsin will stick to the membrane and your digest efficiency will be very very low.
For PVDF, you can use a very dilute Zwitterionic detergent, as described in the publications I have attached. After the in-situ digestion, you can remove the peptides and use a clean-up step to remove the salts etc.
Let me know if you have questions. These protocols work very well; we used membrane digested proteins to characterize proteins for many projects..
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