An antibody recognized NKCC1 at a 75 kDa band, but NKCC1 is usually described between 150 and 200 kDa
Could the protein be cleaved by a protease? Was this reducing blot - could that have cause a different mobility? It depends on the sample and sample prep.
Is there any possibility that the 75kDa is the subunit of the protein ? I wonder if non-denaturing WB could solve this problem if the multimeric structure of the protein causes the problem.
Thanks for answering so quickly!
I didn't do the western yet. It's just the different commercially available antibodies, that end up labelling different bands! But you might be right, and I might check the protocol that has been use, and buffer conditions to figure it out.
The predicted mass for your protein is 131 kDa (EXPASY tools). Look the paper from PLoS Biol. 2012 "Regulation of Brain Tumor Dispersal by NKCC1 Through a Novel Role in Focal Adhesion Regulation" On the fig. 2 they show your protein by western I would really complain:
- there in no mass indicated
- the multiple bands are observed in the cell lines, but there is no negative control showing which are the proper one (RNAi)
- the upper and lover area of the blot are shaved
Could be your protein is cleaved after targeting the membrane in order to regulate it's function?
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