phospho antibody detect untreat lane - (Mar/09/2005 )

Hi, everyone,

I used phospho specific JNK antibody (Cell signaling) for westrn blot. But the most case I observed the untreat lane also shown strong band. I did with 5% BSA (4C for overnight) and 5% skim milk at R/T for 1hr). The both case shwon same results. How can I remove my untreat lane band?

hi
for western blots, i usually block the membrane by 30 to 2hours RT with TBS+5% powder fat free milk and incubate with my antibody overnight 4°, antibody diluted in TBS+5% powder fat free milk.

But in your case did you try to dilute little more your antibody?

Fred

Is it possible that the way you are handling your samples may be leading to the phosphorylation of Jnk in preparation and therefore your sample is fully phosphorylated even in your untreated lane??

Scott

Dear Scott,
I think so. I can not discriminate between untreated group and treated groups. Do you have any solution for this problem?

Wan

Hi Wan,

To test to see if your sample is already fully phosphorylated there are a couple of things you could do. The simplest is to include a third lane which after harvesting of cells or tissue you treat with lamda phosphatase, it should remove the phosphate group and show you whether the problem is with your sample already maximally phopshorylated or whether it is a detection issue with your antibody. Other ways include looking at recombinant protein control lanes etc. but I think the phopshatase should be easiest to do.

I'm not sure about Jnk, it is a stress related, volume related kinase is it not?? I used to work on a stress kinase and found from tissue I had to snap freeze tissue in situ to prevent activation from handling. From cells I found there was already a high level of activation in the cells, however, there was not alot I could do about it other than be as quick in processing and as cold as I could stand (absolutely everything at 4 degrees C). I don't know, maybe someone else out there could suggest a better way but if you lyse in ice cold buffer and complete steps ice cold I found that you could keep the resultant phopshorylation down to a minimum.

Hope I could be of some help.

Scott

I appreciate to Fred and Scott.

Dear Scott,

I always used ice cold lysis buffer and PBS, but always same results.
I searched many papers through PubMed, some papers not shown phospho-JNK band in control and another papers shown in control.
And I have another problem, that is expected phospho-JNK band size are 46and 54 kDa. But always my bands are 43 and 46kDa (very strong band) and only shown 54kDa band in high dose of chemical (very faint band).
Is it reliable?

Could you give me suggestion?

Hi Wan,

I probably aren't the person to be asking, perhaps someone out there is working on Jnk could provide an answer.

From your reply you expect to see two bands one at 46kDa and one at 54kDa and basically you only rarely get the 54 kDa band. I presume that these represent different isoforms of Jnk.

Is the phospho site the same on the two isoforms, ie. could the phospho antibody pick up one isoform better than the other?

If you western blot with a de-phospho antibody is the 54kDa present, if not then the problem is not with the phopsho antibody but the expression in your cells/tissue?

Otherwise there could potentially be differential phosphorylation of the two isoforms in your cell type.

If you are worried about the specificity is there any way you can run a positive control of recombinant protein that has been phosporylated by the upstream kinase. Also the lamda phosphatase treatment I mentioned earlier, that will demonstrate the specificity by knocking off the phosphate group it will show you if the antibody picks up de-phoshpo protein.

Sorry I don't know how helpful any of this is. The only other thing I can suggest is to check the literature, do other people have similar problems with the 54kDa band?

Hope some of this is of use.

Regards,

Scott