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Western Blotting Help!!!!


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#1 DigitalFreak

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Posted 28 July 2004 - 08:00 PM

Hello ALL!

Im hoping someone can explain what is going on with some of my data!!!!
We are currently checking cell viability post cell explant from saphenous vein tissue. Our viability data indicates that we are isolating less cells from the tissue as time increases and that these cells in culture are less viable. So to look at specific cell markers we thought it locgical to run our samples on a SDS-PAGE and use Beta Actin as a loading control. Time after time we are loading equal amounts of protein per lane but our cell specific marker is decreasing (which is logical...that is the amount of cells expressing H-caldesmon.....) but our B-actin which should be ubiquitous to all cells is also decreasing at a similar ratio. This doesnt make sense to me as we were hoping to see loss of protein cell-specific protein, only!! We have used BCA to measure our protein concentations in our cell lysates, and have used kits from different manufacturers with similar results. We have used different antibodies, both monoclonal and polclonal. We have also used different H-caldesmon antibodies. Each time with similar results. Could cell death (which we know by our viability experiments) contribute to the decrease in beta-actin (our internal control) despite the loading of equal amounts of protein. By the way, this has also been done with GAPDH and tubulin. I'm at a loss and cant explain this.

PLEASE HELP?!!!

DigitalFreak

#2 kant0008

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Posted 28 July 2004 - 08:25 PM

Hi!
It has been noted, especially recently, that b actin is not really that great a control, its levels change during various cellular events. So no surprises there. You say you tried GAPDH, which is another housekeeping gene. Another one is PBGD. Are you sure you don't have a global shut down of protein expression, like a shock response?

#3 DigitalFreak

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Posted 28 July 2004 - 09:33 PM

Dear Kant0008,

I'm not sure. Its not that there is no protein coming up on my blots....but the control protein is fluctuating with my cell-specific protein. It looks as if I have loaded decreasing amounts of protein....but I know for sure I haven't. How would i check for a shock response ? I'll try anything!!!!

Thanks for taking the time to answer.



Cheers

#4 kant0008

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Posted 29 July 2004 - 07:57 PM

Hi again,
I would try a different housekeeping gene, there are various examples in the literature, we have previosuly used PBGD. Actin might not be too good a control.
if you want to test for shock response try an RT-PCR for one of the heat shock proteins, you might observe a large increase in expression - I have never done this, so don't much about it. Again literature might be helpful. The reason I though of a shock response inthe first place is because you say you observe decreased cell growth rate and viability. Hope thos helps and didn't confuse you even further.

#5 mujan

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Posted 30 July 2004 - 12:17 PM

the data are the data. chances are all sorts of cell death is taking place in your explants. check your explants with cell death markers. can you normalize with cell number instead of total protein? can you digest the explants, FACS live cells specific for your marker, lyse a known number of cells, then blot?




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