Hello everyone,
I'm trying to detect MUC5AC protein by Western blot. Unfortunately, there was only appears unexpected non-specific bands (picture as attachment).
My experimental protocol as following:
1. Seeding 4x105 A549 into 6-well in culture medium for O/N (next day will reach 60% to 80% confluence)
2. Starvation of cells by SFM medium for O/N.
3. Wash A549 with cold PBS
4. Lysis cells with 60 μl RIPA buffer on ice for 5 min and collect cell lysate by scraped.
5. Froze at -20°C for O/N.
6. Centrifuge cell debris at 14000 g for 15 min at 4°C and collect supernatant
7. Protein concentrations were determined by BCA assay.
8. 100 μg of total protein for each sample was boiled with reducing dye and run 5% SDS-PAGE.
9. Protein was detected by primary Ab (Mouse α Human MUC5AC Ab (Santa cruz, A-5 sc-398985)) and HRP-conjugated anti-mouse Fc secondary Ab.
ps. MCF7 lysate is commercial positive control, beta-actin as loading control
Is there any suggestions, good detection primary antibodies or better protocols to detect this protein?
Thank you very much
Hi, in theory the theoretical weight ~ 36.7 kDa ... you probably got some few pieces around. It can be explained by AB that you use (Santa Cruz is cheap and great but there still so many things) also RIPA is quite aggressive buffer ... also based on your loading controls you are overloaded a little bit a gel (i think). well you can try a different protocol ... use 10 mM PBS instead of RIPA, destroy cell by ultrasonic pulses, use inhibitor of protease (for example from sigma-aldrich). try different primary AB, try pre-cast gel (better gradient one in your case i would say 8-16%). And Wb is also about physics ... Ohm's law and pH .. But again, from my point of view u got just one unspecific band with relatively small density (~35 kDa). You should focus on the band there your controls appears ... and look 5% gel for ~40 kDa protein ... i think, you should try ~ 15 % ... Thats all from advices, you decide. Good luck with your research.
What is the expected weight for the protein, my online sources from the antibody sheet and atlasgeneticsoncology suggest it is a very high mW protein >400 kD. It can be extremely difficult to transfer these high mW proteins, what is your transfer protocol?
Your SC mcf lysate suggest you are transferring high mW proteins but it difficult to ascertain whether you are transferring proteins that are all the way up to 400 kD such as your protein of interest, i.e. perhaps your transfer is effficient up to 350 kD.
http://atlasgeneticsoncology.org/Genes/MUC5ACID41460ch11p15.html
https://www.scbt.com/scbt/product/mucin-5ac-antibody-a-5
However according to abcam the human recombinant Mucin 5 AC protein is approx. 37kD including tags according to abcam. Perhaps your lowest band is the protein of interest, as Grgoriy said, you may get a better seperation/resolve if you used a higher percentage gel.
http://www.abcam.com/recombinant-human-mucin-5ac-protein-ab114218.html
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