I have been trying to look for my protein of interest (phosphorylated PKR) in my western blots and I have been having trouble because there appears to be a lot of backgrounds. Sometimes, I can see what sorta looks like bands but it is hidden due to the background. This is my protocol that I use:
-Prepare Laemmli buffer 2x plus B-mercaptoethanol
- Mix proteins and Laemmli buffer at a ratio of 1:1
-Load 30ug of proteins from my samples
-Electrophoresis parameters are 90V for 15 minutes and then 140V for 1 hour and 15 minutes.
-Soak PVDF membrane on 100% methanol for 5 minutes
-Transfer parameters are 0.4 amps for 1 hour and 15 minutes at 4C fridge.
-Blocking: 5% BSA with TBST 1X for a final volume of 10mL. 1 hour blocking at room temperature.
-I do no washing after blocking, proceed to adding primary antibody.
-Primary antibody diluted at a ratio of 1:1000 in 2% BSA with TBST 1X for a final volume of 10mL. Overnight incubation at 4C.
-Save the excess of the antibody for future use.
-Washing with TBST 1X for 3 times at 10 minutes each wash. The washing volume is approximately 10mL
-Add secondary antibody (anti-rabbit conjugated with HRP) at a ratio of 1:10,000 in TBST 1X at a final volume of 10mL. Incubation for 1 hour a room temperature.
-Washing 4 times with TBST 1X for 10 mimutes each. The washing volume is approximately 10mL.
-Prepare the peroxide buffer and luminol at a ratio of 1:1, in the dark, for a final volume of 1mL. Then add the 1mL to the membrane and quickly proceed to reveal the bands.
-Results: failure. Too much background.
Primary antibody is from Abcam
Secondary antibody is from Sigma. Sigma states that the concentration of the secondary should be 1:80,000 to 1:160,000 however, I found this too small so I went with 1:10,000. Is this why it is failing?
The problem may not be high background. It may be lack of signal. If you expose the membrane for chemiluminescence detection for a long time to try to detect bands that aren't there, it will bring up the background. Consider the quality and concentrations of your samples, your 1o Ab, and your 2o Ab-HRP conjugate as possible sources of the problem. Try to find a sample that you can run as a positive control, such as 1 µg of the purified antigen.
@Adam B Shapiro
Thanks for answering. When I performed the extraction and quantitation of my protein samples, it resulted in the range of 300ug to 600ug of total protein. I thought that would be enough since I only load 30ug of proteins for electrophoresis.
As Adam suggested, include a positive control. Further, try to block the membrane with 5% Non-fat Milk and increase the dilution of the secondary antibody. Make a substock of the secondary antibody so that it will be easy to get the required concentration.
I think this may be a simple background issue. If you are using the conjugate at up to 16X the recommended dilution, I would expect background. It also looks like bubbles may be in contact with your membrane, which is why you can see defined black blotches. I suggest that you take a series of strips of membrane and soak them in tubes with blocking buffer and then add a serial dilution of conjugate from 1:10,000 (current) up to the recommended dilution. Wash and place of film and add substrate. Look for the highest concentration that doesn't generate significant background. That's just a suggestion for troubleshooting.
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