My target protein is low molecular weight membrane protein (LC3 II). It comes out good in cell systems but not in animal lung tissues (white buffy layer on the top of the protein supernatant, cannot be separated using pippette, interferes during WB). Also, I have tried ultrasonication and protein precipitation using acetone but did not work. Any suggestion in providing protocol for protein isolation without lipid contaminants from lipid rich tissues or advise commercial kit is appreciated.
Can you do any kind of classical protein purification, like Gel filtration, ion exchange etc? Usually this gets rid of lipids quite efficiently. In your case, if you just want to remove the optically visible white layer, a simple PD 10 desalting column might be sufficient, the white layer should get stuck on top of the column.
Hope that helps...
Bjoern
Do you think your protein would handle a cold ammonium sulfate precipitation? Most of the proteins I work with handle this better than acetone precipitation or trichloroacetic acid precipitation. The lipids usually end up on top of the liquid layer after being in a refrigerated centrifuge (3,500 - 5,000 x g) with the protein in the pellet. Resuspension with buffer followed by dialysis results in native protein. (When you pour off the supernatent you might need to wipe down the interior of the tube with a Kim wipe to get rid of any residual lipid.) Good luck!
I use a kit from Pierce that separates hydrophobic from hydrophilic proteins.
Hi all... thank you very much for your valuable suggestions!
@ Bjoern... in my lab there is no set up for protein purification. However, PD10 desalting columns idea seems simple to me. My protein supernatant with white fat (lipid) layer on the top will be around 200 ul. I tend to use low volume to get good concentration protein sample. Will I be able to retrieve whole volume except white lipid layer just by gravity? or can I use simple bench top centrifuge for the above columns? Can you suggest any good company source for PD 10 desalting columns please?
@Christopher... i tried acetone and TCA precipitation but didnt work due to hard pellet formation which I am unable to redissolve.
@ Francisco... I have also thought of using heavy detergents... but if possible, I would like to use the same protein sample for IP as well.
@ Maggie... thanks, I heard Pierce kit is good for doing IPs... if time permits, I will try...
Are you talking of this protein?
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC305793/
Hi Nabil... I cannot get the protocol from pubmed... could you specify the PMID please.
@ Paola: Yes, thats the protein I am talking about... do you also do LC3 II studies?
Can you open the file cdd565? No I do not study LC 3 II
This is the corrigenda of the paper published later
"LC3, a mammalian homolog of yeast Apg8p, is localized in autophagosome membranes after processing. Kabeya Yukiko, Mizushima Noboru, Ueno Takashi, Yamamoto Akitsugu, Kirisako Takayoshi, Noda Takeshi, Kominami Eiki, Ohsumi Yoshinori, Yoshimori Tamotsu The EMBO Journal. 2000;19:5720–5728.
In the Materials and methods section of this paper, the amino acid sequence of the synthetic peptide that was used for immunization to prepare antibody against LC3 N-terminal region should be PSEKTFKQRRSFEQC, not PSDRPFKQRRSFADC as shown in the published paper. The authors would like to apologize for this error and for any confusion caused."
Hi Paola... I can open the pdf file, and thanks for bringing this to my notice.
a little expensive but absolutely amazing to get rid of this fatty layer is to use the Columns from the RNaesy minelute cleanup kit from qiagen. You just put your samples on the column and you centrifuge at max speed for 1 minute. Again, it is a expensive but really efficient.
I'd like to try the magic with RNeasy MinElute columns. Does anybody have a protocol how to use them for delipidation?
Hello, Sureshbabu. Did you remove the lipids from proteins of animal tiussue succesfully? I have the same problem and hope I can get some information from you.
Thanks a lot!
Hi Jiangtao,
The way I remove the lipids from animal tissues during protein extraction is to do couple of centrifugation steps at 4 C. I try to take the clear solution (not the lipid layer on the top) as much as I can. Repeat the step two times to get as much as protein sample. This is most economical and works for me. If the lipid layer is too much then increase the volume of the protein lysis buffer and follow the above steps and later concentrate the protein solution if necessary. Hope this helps.
You can simply use Soxhlet extraction apparatus for lipid removal.
- Badges
- Science method