Hi!
I am currently comparing wild type and knockout mice tissues and for the knockout confirmation I am doing western blot and using X-ray film for detection. The problem is that after the development of the film I have weakly visible bands and a strong background. I tried to change the concentration of primary and secondary antibodies, to increase concentration of blocking milk, more washing between primary and secondary and did get some improvement but the film (after developing) is still not as good as it should be. Since I ran out of ideas I would be very pleased with any other solution or idea for this problem?
- protein concentration 20-30 µg/µl
- 12,5 gel SDS PAGE
- blocked for 1 hour 10 % milk in TSBT
- primary (1:1500 in 5% milk in TBST) overnight at +4°C
- wash 5x / 5min in 10% milk in TBST
- secondary (1:5000 in 5% milk in TBST) 1 hours at RT
- wash 5x \5 min with TBST
- chemiluminescent substrate 1:1 ration (2-3 min incubation)
- X-ray film 3-10 min incubated in cassette (even tried overnight)
maybe incubation instead of 4°C overnight, 1h at RT ?
why do you have 10% milk in washing buffer after primary AB?
Please upload some picture of your blot... Without seeing it.. It is difficult to get the actual idea about your problem
Hi Grazia
Well, there are some points that you can work on it. You have 2 problems: background and poor signal
First, Background: you are using skimmed milk (SM) in too high concentration. Tried to use around 3 - 5% as blocking in TBS without Tween. Dont use detergent in the blocking step!! If it does not result, try other blocking agent such as 1%BSA in TBS. Blocking time 1h, 37oC
Perform the washing steps under shaking if it possible
I would add a washing step after blocking (1x/3min).
I agree with Erman and I would remove the SM in the washing solution.
Second poor signal: As solution for prepare both primary and secondary Ab use TBS containing 0.05 -.1% of Tween and 1% SM.
The appropriate dilution of both primary and secondary Ab depends on their concentrations. As rule thumb 1-10µg/ml is enough.
Substrate and exposition time depends of the reagents quality and film but if you resolve the previous is easy to adjust this point.
Good luck and let me know any additional question, Oscar
Amended on 14 August 2018
I have to sadly and repeatedly declare that notorious Wester blotting method and Immunological method are not applicable to hydrophobic proteins (my unpublished observation at the University of Tokyo, Tokyo, Japan: and please see file; IEF for Hydrophobic Protein). I have kindly informed that ELISA method to assay Fucoidan (sulphated poly-L-Fucose) is applicable to urine, but not applicable to serum (kind information from a reseacher at Graduate School of Health Sciences, Gunma University, Maebashi, Gunma, Japan in 2014).
Hydrophobic protein is defined by us that hydrophobicity larger than 52.5%.
Hydrophobicity (%) is defined as
Gly/1.5 + (Cys + Met) x 1.5 + Ala + Val + Pro + Tyr + Phe + Trp + Leu + Ile (%).
Western blot has been reported by Dr. W. Neal Burnette (Fred Hutchinson Cancer Research Center, Seattle, WA, USA) in Analytical Biochemistry, Volume 112, Issue 2, April 1981, Pages 195-203 by using Murine leukemia virus antigen of Glyco-Gag protein/Gross cell surface antigen. This membrane protein has exceptionally low hydrophobicity of 49.6%, which is a level of soluble protein as assessed by our method.
Therefore, another methods are necessary to chemically determine the object protein.
1) please separate the protein by our HPLC-Surfactant-SEC method (please see file; SEC column 300A silica).
2) please quantitatively determine the fractioned protein peak by the Edman degradation method (please see file; HepG2 fucoidan). Please use a reliable Shimadzu PPSQ protein sequencer.
I have 10% milk in washing buffer after primary AB because it is in the protocol I have (was not sure if I can make a change from a solution with milk to milk-free solution since I have primary and secondary AB in milk). But I can try to wash without MS. Definitely will try to decrease MS % for secondary AB and wash after blocking.
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