I have been trying to detect APC (adenomatous polyposis coli) from RIPA lysates of HEK and SaOS2 cells by western. However, I am not able to detect the protein (Mol Wt ~300kDa). I have tried loading up to 150ug protein and transferring at 20V for 26h at 4 degrees in a transfer buffer containing 380mM glycine, 50mM tris, 0.02% SDS. I have also tried immunoprecipitating APC from 500ug of lysate using Ab-5 antibody for 1h at 4 degrees but cannot get any detection. I have been using Ab-1 antibody from Calbiochem, but the antibody is more than 2years old so I do not know if their is any problem with the antibody. Anyone with experience, your suggestions are welcome. Also could you suggest a good antibody.
Just for my interest, did you check the protein lysates with soluble and insoluble fraction? may be your protein is aggregated already and no more available in soluble fraction.
about the wb: did you check the transfer by ponceau staining? I see that in the transfer buffer you don't have any methanol (usually till 20% for high mw proteins).
without this check (ponceau) you could have the same problem with the immunoprecipitation (transfer)
Do the following to make sure your experiment worked: Is the protein of interest present in the gel? I usually run at least two lanes, one for staining and one for blotting). Did you stain the blotted gel with Coomassie to see if the protein transferred out of the gel? Did you stain the blot with Ponceau Red to make sure the protein transferred? Does your blotting buffer contain 15 to 20% methanol? did you check the pH of the glycine buffer to make sure it is is 8.6 or higher? Or use MOPS buffer.
Hi Neeraj, it sounds like the above suggestions could really help, but if you do not find a suitable antibody, we select DNA aptamers as antibody replacements. We have a promotion going if your protein is related to cancer. Please see www.BasePairBio.com
Here are the possible reasons and relative solutions for no western blotting signal.
(1) Not enough transferred protein--The protein expression level may be too low, so just increase the volume of loaded protein; use a positive control; make sure that the lysis buffer you used for sample preparation was strong enough to break the cell wall or membrane, and have included appropriate protease inhibitors; check that if the separated proteins have successfully transferred to the membrane by ponceau staining.And also check with the transfer buffer which you are used doesn’t have methanol.
(2) Too low antibody concentration--Reduce the dilution proportion of primary antibody or secondary antibody; quit the re-used antibody and try a fresh one.
(3) Antibody incompatibility--Ensure that the primary antibody you used can well recognize your target protein in the species being analyzed by performing a Clustalw alignment, and also, the secondary antibody must be chosen according to the primary antibody. E.g. if the primary antibody is a Rabbit Monoclonal Antibody, you should use an anti-rabbit secondary antibody.
(4) Over wash of the membrane--Avoid excessive washing of the membrane.
(5) Too much blocking--Excessive blocking makes it difficult to visualize your target protein, so reduce the concentration of non fat milk appropriately or shorten the blocking time.
(6) Inactive substrate--Switch to fresh substrate.
(7) Not enough film exposure--Increase the time of film exposure.
Hi,
I have been working with APC and it appears to be very difficult to get the APC protein after westernblotting. One best solution to get hold of APC full length protein (300 kD) is to avoid stacking gel while loading the samples. I use anti- Ali (1:1000 in 10% milk ) from abcam which is working good. Good luck.
Thank you Mekala. Well I am going in for abcam antibody since I have tried santa cruz as well as calbiochem. I run 6% resolving without stacking.
Dear Mekala,
Can you also provide me with what transfer conditions you are using and how much amount of protein do you load on the gel.
Thanks.
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