I am having a problem lately. I can't obtain enough protein from cells. I am working with different cell lines and from 1.000.000 cells, I usually obtain 5-10 mg/ml of protein and that is a very low concentration. I used the standard RIPA buffer protocol, plus protease and phosphatase inhibitor. I tried to sonicate, to leave in ice for longer but nothing. Could anyone help me by sharing his/her tips for protein extraction based on cell lines? I never experience any problem with tissues.
Hi - I agree with the above that it is very cell line dependent and also how confluent the cells are at time of lysis… I work with cancer epithelial lines, you say you have 1 million cells which is about the number of cells in a 80-90% confluent well of a 6-well dish, which I would lyse in a minimum of 200uL and would not expect more than 5 mg/mL - if I was lucky - so I'm surprised you think 5-10mg/mL is a 'very low concentration', I think that's huge…..! RIPA buffer should be more than enough, sonication is excessive [unless you are doing CHIP assay, maybe] and for me tends to lead to a lot of lipid contamination (cloudy crap at the meniscus after centrifugation, very annoying). HIgh salt (1M NaCl) to your RIPA may help. You mention 'tissues' at the end of your question, which makes me wonder if you are talking about lysis/extraction from xenograft-like excisions (and which give very high protein con.) and is quite a different question…. Sorry I can't be of more help...
Hi Francesco. I found that you can use lower volume of extraction buffer if you shear the DNA by repeatedly drawing the extract through a syringe with a high gauge needle.
Dear Francesco. There are several points to this. Not knowing which protein you are working with, it is not too trivial to sugest something. Howerver, you should check expression conditions. Perhaps you tweak induction, growth condition or maybe optimize through choice of cell lines.
Next, RIPA is a fine buffer, but generally, try different detegents one by one since each protein requires seperate testing of detergents. After that you might see to sonication and incubation times again.
Any further info you can/may share with respect to the protein could help to better answer your question.
Good luck!
Hi - I agree with the above that it is very cell line dependent and also how confluent the cells are at time of lysis… I work with cancer epithelial lines, you say you have 1 million cells which is about the number of cells in a 80-90% confluent well of a 6-well dish, which I would lyse in a minimum of 200uL and would not expect more than 5 mg/mL - if I was lucky - so I'm surprised you think 5-10mg/mL is a 'very low concentration', I think that's huge…..! RIPA buffer should be more than enough, sonication is excessive [unless you are doing CHIP assay, maybe] and for me tends to lead to a lot of lipid contamination (cloudy crap at the meniscus after centrifugation, very annoying). HIgh salt (1M NaCl) to your RIPA may help. You mention 'tissues' at the end of your question, which makes me wonder if you are talking about lysis/extraction from xenograft-like excisions (and which give very high protein con.) and is quite a different question…. Sorry I can't be of more help...
What volume of extraction buffer you use for the 1.000.000 cells? I completely agree with Richard Elliott - 5-10 mg/mL in general sounds to me as a quite high yield..
I do not know your steps to sonicate samples,
but avoid sonication if you do not intend to break membrane and work with membrane proteins
I agree with the awnsers above. But if you need more protein, very simple: use more cells. If I need al lot of protein for IP, I make protein lysates of 50 up to 200*10^6 cells. When you work with cell lines, you can grow as much a you need.
(And depending on you're protein of interest you could also try a NP40 lysisbuffer.)
Hi there, thanks to everybody I didn't receive any notification from Research Gate so I am seeing your suggestions right now. Yes I usually obtain 5-8 mg/ml from a full confluent flask of COV434 cell line ( a metastatic granulosa cell line). I realised that using double of the protease inhibitor (and DTT) in the extraction buffer (RIPA) improves the protein yield significantly. For some reasons, I expected more from a flasks 77mm2. But consedering your feedbacks I think my yield is considerably good. Sonication is something I usually perform using a bath sonicator more gentle on cells compared to the probe sonicator which works on high frequencies. Thanks again to everybody.
Hi! I do not want to create a separate topic so I ask here.
I am working with Huh7 (a well differentiated hepatocyte derived cellular carcinoma cell line) and when I am isolating proteins I centrifuge the lysate (10 000rpm, 10min, 4C) I obtain very thin, white layer of "something" on the surface of the supernatant. It looks like fat or I don't know...
Did anyone have the same problem? Because I can not calculate the concentration correctly when I am trying to aspire only supernatant I always take a little bit of this "thing".
I used RIPA with 1%Protein Inhibitor Coctail 1%EDTA 1%DTT and 0,1% PMSF.
Thank you for any help!
I believe that you need a specific lysis buffer to optimize protein isolation from liver and/or hepatocytes derived cell lines. See if you can find any reference in the attached file.
RIPA always causes altered protein profile and endogenous baseline due to loss of proteins non-proportionally. Strong lysis buffer should be used for complete lysis. The stickiness can be removed by using a spin column with a special filter. Sample can be ready in just a minute. Try to search Minute Protein extraction kit on Google.
- Badges
- Science topic