Dear expertise,
1) I have some trouble to get reproducible results of phosphorylated protein from western blot. Please give me some ideas and suggestions to get the perfect/reproducible results from the blot.
2.) How different is the western blot protocol for phosphorylated protein from regular western blot?
Thank you in advance for your help.
Please quote your problems in the reproducibility of your experiments
You could try using stain-free gels for total protein normalization and fluorophore conjugated secondary antibodies in infra-red spectrum if you desire more reliable quantification.
In terms of the general advice, do not use skim milk as the blocking/antibody diluent agent, it contains phosphatases that ruin most phosphorylated proteins on the blot exempt most abundant one during the first incubation. The best results I had with BSA 1-2% it is also allowed to reuse primary antibodies for months where skim milk degrade after several weeks.
Otherwise, I agree with other researchers you need to provide more information regarding your target, experimental conditions (lysis buffer, protease/phosphatase inhibitors, gels, transfer conditions, etc) and issues you are facing to get a meaningful answer.
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