Hi,
I'm writing my master's thesis on the characterization of human proteins which I express and purify from E. coli. I'm performing western blot to verify my protein, but the protein standard signal is very week.
I used the BIO-RAD kit for making 12 % SDS-PAGE gel and loaded 5 µl of Precision Plus Protein Western C standards (stain-free). I blotted the membrane using the trans-Blot turbo system with 3 min transfer time and blocked with 5 % BSA PBST. I used anti rabbit primary AB 1:2000 for my protein, then washed 3x5 min with PBST and applied secondary Goat anti rabbit-HRP IgG 1:2000 together with Precision Protein Strep Tactin-HRP Conjugate 1:5000 to detect the standard. Then washed 3x5 min with PBST and applied 2 ml substrate solution (BioRad Clarity Western ECL Substrate) in a 1:1 ratio.
The detection of the protein works well, but the standard signal is too weak. Does anyone have any experience with this? Any advice is very much appreciated.
Hi Celine,
may you cover the sample area on your blot with a peace of paper while imageing. The imager will accumulate the signal of your protein standard until you can detect something. In the end you can merge the image of your samples with the image of the protein standard. But if you used a colormetric standard you have to check the settings and merge then the colormetric protein standard with the flourescence image of the samples.
kind regards
David
David Oguama suggestion is good if you maintain good alignment. Alternatively, you may try to decrease PBST concentration or duration in the last washes as I had this problem before. I also added bit more standard. The timing of exposure and duration of developing the film may also contribute. try loading less sample so the film does not become unbalanced in signal intensity and blotchy which greatly limits the sensitivity of the developer. As for your current developed films, perhaps scan the film and use signal intensity measuring software like Adobe or imageJ to identify the faint standard bands.
Hi:
Could you directly get the standard marker by white light and merge the two images (HRP and standard) by photo or other software?
I agree with Rabeah Al-Temaimi that more standard or editing the image will give the result that you want and those are probably the best tries. If you are trying to improve the signal on old images then it is possible to intensify the film signal using Kodak chromium intensifier which is just acidified potassium dichromate then re fix the film. This can be repeated a few times to increase weak signals as the chromium builds up on the silver image. If the size standard is at the edge of the film then manual intensification could be used selectively to intensify the size standard image
Thank you for all of your nice suggestions. I decreased the duration of the last wash suggested by Rabeah Al-Temaimi, and it worked very well. Thank you so much.
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