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western blot lysates

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western blot lysates

Postby nunyfra » Mon Apr 16, 2007 1:46 pm

Hi!!

Who can help me???? :oops:

My problem regards cellular lysates of primary human lung mast cells.

I am investigating the expression of a protein in these cells by western blot. I didn't see anything in the lane corresponding to these cells. Of course this is not a problem since there is the possibility that these cells do not express it. Howewer, I didn't find GAPDH. this happened twice times although the second time I used the half of lysis buffer for the same number of cells (I noted that the lysates were more similar to a gel consistence than the first time). I used the same lysis buffer also for LAD-2 and HMC-I (2 mast cell lines) and I had not any problem.

Is it possible that the protocol of purification of mast cells (positive immunoselection with anti-CD117 antibody) may damage mast cells in any way?

Thanks a lot for your help!!
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Postby blcr11 » Mon Apr 16, 2007 8:38 pm

This is an double-antibody, colorimetric detection system? (alkaline phosphatase- or catalase-conjugated anti-Fc antibody, or something like that). I would be suspicious of the gel-like consistency of the lysate. That could be gross contamination by chromosomal DNA. I don’t think that would impair the binding, but it might make it difficult to add consistent amounts of sample to the gels. All that gloppy stuff plugs up the pipet tip. You can add some DNAse to get rid of that problem, if you’re having one in that department.

I don’t know the protocol for CD117 immunoselection, but unless it also involves proteolysis at some point, the extraction is probably OK (baring silly things like omitting detergent, etc.) My guess would be that your AP-Fc antibody (or whatever enzyme it is) has gone bad, or been overdiluted, but that’s just a guess.
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Postby nunyfra » Tue Apr 17, 2007 5:01 am

Thank you for your considaration!!
Probably I made a mistake in my explanation.... :oops: :?
The anti-CD117 antidoby is conjugated to magnetic beads. It is incubated with all the cells, then the suspension is pushed through a column that capture mast cells. It serves to me to purified human lung mast cells from the other ones. At the end I washed the cells and I counted them (0.5x10^6). The purified mast cells were lysated with 20 mcl of lysis buffer and the total amount of proteins were evaluated (4,5 mcg/mcl). I loaded 35 mcg/lane. (7.7 of lysate + sample buffer + 2.5% beta mercaptoehtanol)

You are right about DNA contamination and I will assess the possibility to add DNAse.

P.S. I made this experiment twice, the result was the same even if the first time the gel consistence were not present (The cells were about 1.5x10^6 and they were lysated with 90 mcl of lysis buffer; total amount of proteins: 1.3 mcg/mcl)
:o
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