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No proteins after HeLa cell lysis


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#1 soumya.katamani

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Posted 21 August 2013 - 03:44 AM

Hello,

 

Ive been having a weird problem with my cell lysis lately. I lyse my cells, I quantify my proteins and load around 50ug sample on 8% gel and after transfer I stain my nitrocellulose membrane with Ponceau.... I don't see any bands on my membrane and I reveal the same membrane and see no band...

 

to elaborate,

I use the following lysis buffer

Tris - 50mM

NaCl - 100mM

EDTA - 5mM

NP40 - 1%

 

I used regular HeLa cells (75cm2 flask) and scrape them in 500ul LB (+Protease inhibitors) after letting them on ice for 20 min. then I cfg the samples at 10,000 rpm for 10 min. 

I collect the supernatant and quantify using Bradfords rgt. I generally get an OD of 0.1 - 0.2 which seems ok. then I approximately load 50ug protein sample on an acrylamide gel. after the transfer I see the ladder clearly on the membrane (which means my transfer was alright) and then when I stain the membrane using ponceau, I see nothing. I reveal the membrane anyways and I see noting.

 

I don't understand where my proteins went missing!!!!!! 

 

There is no problem in running and transfer coz I loaded just the cell media and I see bands clearly upon transfer and then when I stain the gel with coomassie I see nothing in the gel as well.

 

So, I guess the problem is somewhere in the lysing step.

 

Kindly, help....



#2 Curtis

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Posted 21 August 2013 - 08:31 AM

Why don't you stain a gel before transfer?

#3 bob1

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Posted 21 August 2013 - 12:54 PM

If it is a normal bradford assay, then it isn't compatible with that much NP-40 (assuming it is NP-40 used, not nonidet-P40!), you need to use 0.25% or less, which could be giving you the strange readings. 

 

It could also be that your kit is old and the reagent has gone off.  What do your blanks look like?



#4 Inmost sun

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Posted 21 August 2013 - 12:58 PM

Are you doing SDS-PAGE or native PAGE? for SDS-PAGE, samples must be tretaed with SDS sample buffer (Laemmli buffer) before transfering to the gel






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