Problem on MAPK SAPK/JNK detecting - (Mar/07/2006 )

Hi everyone,

I've been working on western blot with MAPK p38, SAPK/JNK, and Erk1/2 from Cell Signaling. I have no problem of detecting the p-38 but I sometimes got weak signal on Erk1/2 and I never detect the SAPK/JNK on both phsopho- and non-phospho-linked. I am wondering if any of you detect the SAPK/JNK MAPK successfully. Help!!! Please..............

Scarlett

---

hey Scarlett

when we've looked at phospho/non-phospho westerns, we have indeed gotten all 3.

we got our p38 from cell signaling, and our erk 1/2 and jnk from promega

it did take more extract to get a clear picture of phosphorylation for the erk and jnk, but they both worked...we used the non-phosphorylated as sort of a positive control because it always worked

what have you tried? what controls have you done?

---

Hi

Thanks for reply so soon. I used the SAPK/JNK from Cell Signaling and I used the non-phospho SAPK/JNK as control. However, none of the blot I did before come out with any bands even on the controls. I tried double the concentration but no bands still. I just finished 4 blots this afternoon and only one of them works but signal still very weak. So, what should I try next? Thank you very much

---

no, I mean positive and negative controls...see what I mean? and have you done anything to optimize your antibodies? like, for example perform a few dotblots with varying primary and secondary antibody concentration, that sort of thing, so you can achieve a good protocol without beating your head against the wall spending a bunch of time and money doing the whole thing...or have you already tried all this?

---

Hi aimikins,

Sorry about the delate of the reply. I've been busy doing the blots.

Well, we used normal saline as negative control and we used LPS and MALP-2 for positive control. However, we test for three different cell type, THP-1(human macropages), RAW(mouse macropages) and XS52 (mouse DC). So far, I only have the p-JNK bands on MALP-2 on THP-1 cells which are a very faint ones. So, any idea we should try or any suggestion for a better positive control. Besides, the p-ERk1/2 and p-p38 all came out very good on the same membranes (after strip) in all cell types we tested.

Hope to hear back from you soon. Thank you very mcuh

Scarlett

---

Hi there,
I have been looking for a good protocol for making a total protein extract from Drosophila adults. Are you making your protein from Drosophila. If you do. Would you mind sharing your protocol with me. I am running out of time for my presentation and need to do some westerns.

Thanx in anticipation.
Help me out Pleaaaaaaaaaase
Bilal

---

Hi Bilal,

Sorry, we did not make our protein from Drosophila.

good luck

Scarlett

---

hmmmm

JNK should be phosphorylated by LPS in a dose-dependent manner, mostly; that is what we have found in our cells. I have never used MALP2?

I am assuming you have tried a few different concentrations, and what induction timeframe do you use? we have seen induction within 15min and it persists for a while


what is your detection method? I am thinking the main problem should lie somewhere with the antibody dilution or detection method; it doesn't really fit that the problem would be induction since you are getting p38 and ERK to show up

---

Well, we used 0',15',30',60'. The MALP-2 that I detected is activated on 15' and 30'. We used Chemiluineces to detected the signal. Besides, the concentration I used for LPS is 10ng/ml and 100ng/ml. And I used 1:1000 dilution for the Ab according to the instruction.

What's bother me is that the p38 and Erk works pretty well but the JNK doesn't work. I did the p-JNK first before testing the p38 and Erk(after strip). I don't know if it is the Ab or just it's really not activate.

My boss just find a paper that they used p-JNk form cell signaling too. Their bands are faint too except for stimulated with TNFa. My boss told me to try that next week but I am wondering if it is worth to try.

Please let me know if you have any good idea. Thank you very much. Nice day!!!

---

Thanx anyway man.
Cheers
Bilal

---