It is in the literature that there are multiple Claudin proteins expressed in MDCK cells. Running the lysate in 50ul well SDS-PAGE, then transferring with BioRad Transblot system and traditional plate semi-dry transfer with 4.5um PVDF membrane (trying both for trouble shooting). Protein is transferring as verified with B-actin, but none of the lysates are showing any of the Claudin-family proteins (1:1000 primary and 1:3000 secondary). I have done both these transfer methods plenty of times with proteins of a range of molecular weights and never had this issue. I have remade all the buffers and have had success on other projects in the meantime. Why can I not see any of the Claudins? Are they too small (12kD) and are transferring through the membranes?
Hi, we use MDCK II cells and successfully detect claudins in these cells.
If you use transfer buffer containing SDS, claudins may go through the membrane. I would recommend to omit SDS.
MDCK II cells express claudin-1, claudin-2, claudin-3, claudin-4 and claudin-7. (MDCK I cells do not express claudin-2.) They are all ~22kD and the bands are detected ~18kD in our experimental setup.
For antibody information, see Otani T et al., JCB, 2019 and Saito AC et al., MBoC.
Good luck!
Tomo
Hi, we were able to detect claudins in couple of cell lines. As suggested above transfer buffer shouldn't have any SDS. Also, use higher percentage gels so the bands separate well. Since claudins are lower molecular weight- use 12-15% range acrylamide gels. Certain claudins may require use of primary antibody at 1:500 (depends on the antibody) and secondary ranging 1:1000 to 1:2000.
Hi Penny, I used driblot system and PVDF membrane and seen Claudins from other cell lines. Along with omitting SDS, I would also suggest that you check the concentration of your lysate. Claudins are much lower in copy number if you compare them with beta Actin. If the expression of your desired Claudin is lower in that cell line, you need a concentrated lysates to detect it. I have used at least 20 microgram of lysate samples to get a decent band of moderately expressed Claudins. I have also observed that some common lysis methods are not great for detecting Claudins.
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