Help with protein extract! - (Oct/17/2006 )
Hi!
I'm trying for the last few weeks to make "good" total protein extract from WI-38 human lung fibroblasts, but always concentration is very low, even the cells are growing in 150cm flasks. I have tried different lysis buffers and procedures but still its not enought to perform WB or Co-IP. I have the feeling that I'm not scrapping all the cells from the flaks, maybe Trypsin would help, but then I'm not sure about further applications like IP. Thanks for help.
If you trypsinize the cells you can still perform IP. As far as I remember, WI38 spread on the surface of the flask/dish resulting in a lower number of cells/area than other cell lines, don't they? You could try using more cells if you don't need to transfect them
Hi!I'm trying for the last few weeks to make "good" total protein extract from WI-38 human lung fibroblasts, but always concentration is very low, even the cells are growing in 150cm flasks. I have tried different lysis buffers and procedures but still its not enought to perform WB or Co-IP. I have the feeling that I'm not scrapping all the cells from the flaks, maybe Trypsin would help, but then I'm not sure about further applications like IP. Thanks for help.
Did you use any detergents on your lysis buffers?
Hi!I'm trying for the last few weeks to make "good" total protein extract from WI-38 human lung fibroblasts, but always concentration is very low, even the cells are growing in 150cm flasks. I have tried different lysis buffers and procedures but still its not enought to perform WB or Co-IP. I have the feeling that I'm not scrapping all the cells from the flaks, maybe Trypsin would help, but then I'm not sure about further applications like IP. Thanks for help.
Did you use any detergents on your lysis buffers?
i'm using RIPA buffer with complete potease inhibitor coctail and also phospatase inhibitors.
When its about fibroblasts... I use at least 4 10 cm plate and get enough protein to do.. seeeveralllll westerns.. using Ripa, I know a guy who prepares extracts from 1 (35 cm) well enough for 2 or 3 westerns
Hi!I'm trying for the last few weeks to make "good" total protein extract from WI-38 human lung fibroblasts, but always concentration is very low, even the cells are growing in 150cm flasks. I have tried different lysis buffers and procedures but still its not enought to perform WB or Co-IP. I have the feeling that I'm not scrapping all the cells from the flaks, maybe Trypsin would help, but then I'm not sure about further applications like IP. Thanks for help.
Did you use any detergents on your lysis buffers?
i'm using RIPA buffer with complete potease inhibitor coctail and also phospatase inhibitors.
Hi!I'm trying for the last few weeks to make "good" total protein extract from WI-38 human lung fibroblasts, but always concentration is very low, even the cells are growing in 150cm flasks. I have tried different lysis buffers and procedures but still its not enought to perform WB or Co-IP. I have the feeling that I'm not scrapping all the cells from the flaks, maybe Trypsin would help, but then I'm not sure about further applications like IP. Thanks for help.
Did you use any detergents on your lysis buffers?
i'm using RIPA buffer with complete potease inhibitor coctail and also phospatase inhibitors.
Hmm, how you prepare the extract?. Do you use Trypsin?. Thanks for all suggestions
I use trypsin 
When cells are detached, resuspend with medium to quench the trypsin. then spin down, then pbs washing, then lysis buffer...
Do you have a positive control to check your procedure? maybe something wrong with your buffer...
When I did my first IP, I wash too harsh, all my proteins were gone.. Sometimes operation is also a problem......
I used number of home made lysis buffer for coip expts, had a tough time with it. now i use M-PER mammalian protein extraction reagent from PIERCE for my ip experiments . it lyses both adherent and suspension cells amazingly and is well suited for coips. check it out at their website
rajgene