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It'sNotMe's Content

There have been 3 items by It'sNotMe (Search limited from 16-August 19)

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#154829 Insoluble Protein of Interest Resulted from Mammalian Gene Expression - Can Any

Posted by It'sNotMe on 08 May 2013 - 08:35 PM in Tissue and Cell Culture

Thank you for your reply, that's good to know. For SDS-PAGE, I just resuspend the lysis pellet with sample buffer and boil it for 10 mins until it dissolved. I have indeed tried usual method by denaturing the pellet using 8M Urea mixed with column binding buffer (10mM Imidazole) and also with 6M Guanidine-HCL but the pellet did not dissolve. However when I sonicated the pellet in 8M urea the pellet dissolved. I sonicate for 5 mins. This quite peculiar for me. Can you please advise?

#154762 Insoluble Protein of Interest Resulted from Mammalian Gene Expression - Can Any

Posted by It'sNotMe on 08 May 2013 - 02:30 AM in Tissue and Cell Culture

I am having a peculiar problem of protein of interest being detected in lysate pellet rather than supernatant after chemical lysis (M-PER Mammalian Protein Extraction Reagent). My initial thought was that the protein might have been membrane bound due to the presence binding domains at the C-terminal of the protein of interest. However, I have repeated the extraction using RIPA buffer which also resulted in protein detection in lysate pellet. Correct me if I'm wrong, eukaryotic expression does not result in inclusion bodies as prokaryotes, so it is safe to rule out aggregation and misfolding.

The following are the information of my experiment:
- Cell : CHO-S suspension cell
- Expression System: Invitrogen's Freestyle Max CHO Expression System
- Detection Method: Western Blot

Protein of Interest Properties:
- Tags: 6Xhis and GFP
- Molecular weight: 52kDa
- Theoretical pI: 8.68
- Aliphatic index: 68.88
- Grand average of hydropathicity (GRAVY): -0.415

I am doubtful whether the protein exhibits hydrophobicity with low solubility (contrary to the hydropathicity index) or is it being precipitated by the the lysis buffer. The lysate pellet is green due to the GFP tag, which is also confirmed by fluorescence microscopy with DAPI staining. I have tried sonication with the cell being suspended in M-PER (I also suspected incomplete cell lysis). I would like to repeat sonication by using different suspension buffer i.e. PBS. In the meantime I would eagerly welcome suggestions and insights for my problem.

#134671 Secondary AB blocking

Posted by It'sNotMe on 17 May 2012 - 08:54 AM in SDS-PAGE and Western Blotting

Hi everyone,

I have a simple question, currently I'm having problem with my western blot with multiple bands. So now I trying to optimize it by:

- Overnight BSA blocking
- Decreased primary antibody concentration (5uL in 10mL) and increased in incubation time
- Decreased secondary antibody concentration (5uL in 10mL)

I am interested in blocking my secondary antibody (Goat vs Rabbit) with donkey serum (since its available in the lab). Is it possible since its a different animal?

Thanks for your time and attention..

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