I am using Phospho-SAPK&JNK pThr183+Tyr185 Antibody (E.665.10) of Thermofisher (Pierce antibody). Protocol followed by me is as follows.
1. PMN fixation with 4% PFA/ 30 min/ 4 degree celcius.
2.Coating a layer of PMN on grease free slide followed by dry fixing.
3.Hydration with 1X PBS. Permiabilization with methanol /30 min/ -20 degree celcius.
4. treating it with 0.25% triton-x-100 for 10 min /RT.
5. Blocking 1% BSA.
6.Primary antibody dilution: 1:200 in 1% BSA/ 1hr RT
7.Secondary antibody: 1:250.
Mounting in vectasheild.
By using this protocol I am getting very weak staining. Please suggest me suitable protocol for PMNs.
Two quick things 1) At least in Drosophila, pJNK staining has notoriously been bad for every antibody on the market. So, could just be the antibody
2) If you haven't already, try using TBS as a base buffer instead of PBS- the Phospho Buffered Saline sometimes can disrupt phospho antibody detection.
Dear Ryan,
I will definitely try your suggestion. Should I reduce washing steps?
Because what I observed by reducing washing steps, instead of getting nothing something starts appearing. I have never tried with TBS before. Is it possible for you to share pJNK staining image with researchgate?
I don't have any proper idea how actually it should look like in control cells. i have always seen anisomycin treated images of pJNK.
Please share some images if possible...
Thank you.
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