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IP my protein from culture supernatant


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#1 tasosp

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Posted 06 July 2010 - 04:17 AM

hi all,

Has anybody IPed a soluble protein from the culture supernatants?
Does anyone have a good protocol for this procedure?

I am either looking for the native soluble protein or I transfect 293T cells with a plasmid expressing my protein, and im looking in the supernatant.

thanks in advance

t

#2 Curtis

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Posted 06 July 2010 - 08:22 AM

you want the whole protocol for IP?

I am also doing transfection and IP.

- scrape your cells after 12-24hr post-transfection
- wash with cold PBS 2 times to remove FBS
- Lyse cells with mild detergent lysis buffer (RIPA is risky some times, use 2% CHAPS buffer)
- rotate at 4C for 1 hour, end to end
- centrifuge 10,000 RPM for 10 min, save supernatant at -80C, do Bradford assay to measure protein concentration
- in the mean time add 1.5 ug of your IP antibody to 20ul protein G or any other affinity gel, top up with PBS to 200-500ul, rotate 4 hours at 4C, block with 1% filtered BSA for 2 hours. wash with PBS 2 times.
- add the cell lysate to the beads (2000 ug above of lysate for 20ul protein G, some people use less some use more).
- rotate overnight at 4C
- remove supernatant (save it)
- wash with PBS 3-5 times
- add 20-30 ul SDS-PAGE sample buffer
- boil 95C for 10 min
- run the supernatant for western blot, end of story

remember that it is better to have IP and wb primaries antibodies that are raised in different animals, eg. IP primary raised in rabbit, wb antibody raised in mouse (find the reason why yourself :D ).

Edited by Curtis, 06 July 2010 - 08:24 AM.


#3 tasosp

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Posted 06 July 2010 - 08:48 AM

curtis thanks,
however I need a protocol (if there is one?) to IP a protein not from the cell lysate but from the supernatant of my culture. I want to collect the supernatant of my cell culture and try to IP there my soluble protein using an antibody. maybe thats impossible :D




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