Running cell suspension on western blot for detection of IFN gamma

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Row
Row's picture
Running cell suspension on western blot for detection of IFN gamma

Hi,
I'm trying to run cell suspensions on SDS PAGE and detect IFNy with monoclonal antibody in western blot. My problem is that the suspensions are very sticky/viscous and difficult to pipette. also when loading onto gel they appear to float upwards! The suspensions are in sample buffer (0.5M Tris-HCL, pH 6.8, glycerol, 10% SDS, DTT, Bromophenol blue). Is there a better way to lyse the cells before loading on to gel? Also, when i do western blot I'm getting non-specific binding (using 1% BSA in TBS to block for 1 hour). any ideas?
thanks

protoldo
protoldo's picture
Hola Row: The viscosity is

Hola Row: The viscosity is due to unbroken DNA, so is better sonicate the sample for about 5 s. in a sonicator with a thin probe. After that, centrifugate the sample for 2 mins and apply the supernatant with loading buffer.About blocking I always use 5% powerded milk without cream, dissolved in the washing buffer PBS/TBS 0.2% Tween 20. In your case I would increase BSA to 2%. Good Luck

Row
Row's picture
Hi Protoldo

Hi Protoldo
Thanks for your reply! I just found it. I am trying a different method, by lysing my cells and immunoprecipition of protein of interest prior to running on gel. Fingers crossed-it is a very laborious process.
I will try your suggestion next time!
thanks
row