Hi, this much depends on your antibodies. Also, consider the stripping method carefully: some harsh protocols might lead to loss of target protein (not only the antibody). Sometimes you loss more protein the more you detected during first round (protein is "co-stripped" with antibodies)
You could try the following:
If the epitopes of anti-target antibody and anti-phosphotarget antibody differ and being located at distant sites of the protein, use "mild" stripping and you should easily detect reliable signals. As control experiment: prepare a sample and split into 4 portions, run gels, blot and do the following detection procedures: sample1 anti-target, sample2 anti-phosphotarget, sample3 anti-target/strip/anti-phosphotarget and sample4 anti-phosphotarget/strip/anti-target. Compare the phosphorylation levels from sample2/sample1 and from sample 3 and 4 before and after strip. If the ratios are comparable you are ready for quantification.
Sure. Of course it depends on the quality of the antibody and the strength of signal, which can only be assessed empirically, but we do it for lots of different phospho-proteins.
I detected MKP-1 (DUSP1) using ECL methods, but I also wanted to look at MAPK using the licor fluorescent system. Though PVDFs have a lot of high background ... I'm going to try it anyway and see how it turns out. Thanks everyone!
Hi, this much depends on your antibodies. Also, consider the stripping method carefully: some harsh protocols might lead to loss of target protein (not only the antibody). Sometimes you loss more protein the more you detected during first round (protein is "co-stripped" with antibodies)
You could try the following:
If the epitopes of anti-target antibody and anti-phosphotarget antibody differ and being located at distant sites of the protein, use "mild" stripping and you should easily detect reliable signals. As control experiment: prepare a sample and split into 4 portions, run gels, blot and do the following detection procedures: sample1 anti-target, sample2 anti-phosphotarget, sample3 anti-target/strip/anti-phosphotarget and sample4 anti-phosphotarget/strip/anti-target. Compare the phosphorylation levels from sample2/sample1 and from sample 3 and 4 before and after strip. If the ratios are comparable you are ready for quantification.
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