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WB for phosphorylated protein complex in rat intestinal tissue


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#1 wmw

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Posted 19 February 2009 - 02:06 PM

Hi all,

I would really appreciate some advice on the following:

I am trying to do a WB for a phosphorylated protein in tissue homogenizate from rat intestines and I am wondering about a couple of things:

1. when I sacrifice the animals do I have to worry about dephosphorylation, in other words, how long aproximately do I have after I take out the colon

2. I cut open the colon and use the back of a knife to get the feces out, if necessary I rinse in PBS. Should I maybe add some kind of protease inhibitor to the PBS?

3. after this I immediately snap freeze the tissue, then I freeze at -80. I then weigh the tissue and add 9 parts of 1x cell signaling lysis buffer
1X Cell Lysis Buffer:
20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Na2EDTA
1 mM EGTA
1% Triton
2.5 mM sodium pyrophosphate
1 mM beta-glycerophosphate
1 mM Na3VO4
1 g/ml leupeptin,

should I add any other protease/phosphatases than the ones already in there (and the PMSF that they suggest you add) to prevent dephosphorylation?

5. I've looked at a lot of different protocols but they all centrifuge on different speeds. What should I use to get my protein in the lysate, part of it is nuclear.

Thanks so much for any advice!

#2 bob1

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Posted 19 February 2009 - 03:51 PM

Dephosphorylation is pretty quick, but I would say that you won't have too much problem if you are relatively quick with your freezing <5 minutes should be fine.

Rinsing is good, you shouldn't need to add a protease inhibitor. You could try Ringer's solution instead of PBS, but it shouldn't matter much. It is most important here to get rid of faeces and mucus from the gut.

I would add Complete protease inhibitor from Roche as well, it contains a range of proteases inhibitors in tablet form. It is quite expensive, but definitely more convenient than making up lots of different solutions. Make sure you have an excess of phosphatase inhibitors.

So long as you are grinding your tissue well, you shouldn't have a problem getting your protein into solution. The centrifugation steps are usually to remove big bits of tissue and other insoluble stuff.

#3 jiro_killua

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Posted 19 February 2009 - 04:20 PM

Dephosphorylation is pretty quick, but I would say that you won't have too much problem if you are relatively quick with your freezing <5 minutes should be fine.

Rinsing is good, you shouldn't need to add a protease inhibitor. You could try Ringer's solution instead of PBS, but it shouldn't matter much. It is most important here to get rid of faeces and mucus from the gut.

I would add Complete protease inhibitor from Roche as well, it contains a range of proteases inhibitors in tablet form. It is quite expensive, but definitely more convenient than making up lots of different solutions. Make sure you have an excess of phosphatase inhibitors.

So long as you are grinding your tissue well, you shouldn't have a problem getting your protein into solution. The centrifugation steps are usually to remove big bits of tissue and other insoluble stuff.


about the protease inhibitor, Roche tablet is very expensive and I would suggest the inhibitor cocktail from Sigma, cheaper alternative




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