Hi everyone! I'm very confused and desperate with a problem I'm facing with my Western Blots. I'm trying to quantify CX3CR1 and CCR2 in skeletal muscle, liver and spleen from SOD1G93A mouse model.
My problem is that though I see this apparently correct Ponceau Staining after the tranfer, which led me to think that the proteins migrated correctly, in the developed film I can't see anything but a protein blob in the upper part of the membrane (sorry for the bad quality of the developed film, I took the photo with my mobile phone). What could be happening? The film suggests that my samples didn't migrate correctly but if this is the case, why is the Ponceau OK?
My general protocol is:
- Protein extraction with RIPA buffer
- Genomic DNA elimination by passing through a 25G needle (I had problems with genomic DNA at first, which I thought was the cause for the "bad migration")
- Boiled samples at 98ºC for 3 min before loading
- Loaded 25 micrograms of protein per well, stacking gel at 4%, separating gel at 12%
- Electroforesis at 120V until I got a nice protein separation (apparently)
- Tranfer at 400 mA for 75min
- PVDF Membrane Blocking with 5% Skimmed milk TTBS, for 1h at RT.
- Incubation with primary antibody in blocking solution (CX3XR1 and CCR2 from Santa Cruz, 1:1000), overnight at 4ºC
- 3X, 10 min washes with TTBS
- Incubation with secondary antibody in blocking solution (1:5000) for 1h at RT.
- 3X, 10 min washes with TTBS, incubation with HRP sustrate and film developing in dark room.
Any suggestions or ideas will be much appreciated as I'm really desperate and I keep getting the same results everytime and with each one of the three tissues selected.
What are your loading buffers and running buffers? Your staining doesn't lie. Your proteins are migrating. However, you may not be denaturing samples enough, so the proteins of interst are still bound to something with higher MW. I always heat for at least 5 min at 100C, sometimes more, and I have SDS in loading buffer and running buffer.
Are you trying to use both antibodies at once on the same membrane? I would advise against that until you get bands with them individually. Are you sure your antibodies work and are not expired? You may be getting bands from something with homology, but not the correct target. The strongest bands on the membrane are not always the right and most specific bands--that depends on the antibody specificity. Have you tried using stronger substrate or for longer exposure time to see if you also have faint bands at the right size? If thise doesn't help, I suggest trying an antibody from a different company.
Dear Samanta,
Don't feel bad. Biology benchwork always drives people crazy. For your problem,
Thanks everyone for your advice! Finally I found out what the problem was. As my two target proteins are membrane receptors, the boiling before loading to the gel wasn't helping at all. My target proteins seemed to be agregating and not entering the separating gel. That's the reaseon why all the other proteins were correctly separated but my target proteins did not appear anywhere. If anyone is facing this problems try not to boil the samples and simply load them with the loading buffer. In adittion, running the samples at 60V for 20 min and then at 120V until the end seemed to help too. The bands are not very intense but I was expecting that and they are just fine for quantification.
As I said, thank you for your help!
Best regards!
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