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PCNA (proliferating cell nuclear antigen)


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#1 moljul

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Posted 17 June 2010 - 02:12 AM

before I am establishing proliferation assays (BrdU...etc) for our new laboratory, i want to know, if detection of e.g. PCNA expression is an adequate measurement of proliferation?

thx for any recommendation

#2 rkay447

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Posted 17 June 2010 - 09:28 AM

Although the expression level of PCNA does fluctuate in a cell cycle regulated manner, in my studies when I would stain asynchronous populations for PCNA and detect by IF, I saw strong nuclear staining of all cells and it was impossible to determine reliably which cells were expressing higher than others. However, if you permeabilize cells before fixation, you wash away PCNA that is not chromatin-bound. This is a strong indicator of cells in G1 and S phase!! I had great results with a 5-8 minute permeabilization in ice-cold CSK buffer followed by one wash and methanol-fixation. Abcam offers both mouse and rabbit antibodies to PCNA that are very nice for IF. BrdU, however, is the most commonly used and accepted method for identifying S phase cells. Invitrogen offers a new analog, EdU. The detection of EdU incorporation is not antibody based and therefore does not require DNA denaturation but a major drawback is that this system is not compatible with fluorescent-tagged proteins or phalloidin so if you want to look at the S phase of cells expressing a GFP/YFP/RFP-tagged protein, you have to use BrdU. The big problem is that the acid denaturing step for the BrdU destroys the GFP molecule (AND epitopes so antibodies don't work!!). It took me a long (LONG) time to work it out but you have to stain the GFP/YFP/RFP with antibodies and then do a second fixation before doing the acid treatment. Let me know if you are interested and I can send you my protocol.

#3 moljul

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Posted 18 June 2010 - 12:41 AM

Although the expression level of PCNA does fluctuate in a cell cycle regulated manner, in my studies when I would stain asynchronous populations for PCNA and detect by IF, I saw strong nuclear staining of all cells and it was impossible to determine reliably which cells were expressing higher than others. However, if you permeabilize cells before fixation, you wash away PCNA that is not chromatin-bound. This is a strong indicator of cells in G1 and S phase!! I had great results with a 5-8 minute permeabilization in ice-cold CSK buffer followed by one wash and methanol-fixation. Abcam offers both mouse and rabbit antibodies to PCNA that are very nice for IF. BrdU, however, is the most commonly used and accepted method for identifying S phase cells. Invitrogen offers a new analog, EdU. The detection of EdU incorporation is not antibody based and therefore does not require DNA denaturation but a major drawback is that this system is not compatible with fluorescent-tagged proteins or phalloidin so if you want to look at the S phase of cells expressing a GFP/YFP/RFP-tagged protein, you have to use BrdU. The big problem is that the acid denaturing step for the BrdU destroys the GFP molecule (AND epitopes so antibodies don't work!!). It took me a long (LONG) time to work it out but you have to stain the GFP/YFP/RFP with antibodies and then do a second fixation before doing the acid treatment. Let me know if you are interested and I can send you my protocol.


thx rkay447 - i think iŽll try the EdU (Invitrogen) sounds good!!! ^_^




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