problem in membrane protein isolation

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abcdef
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problem in membrane protein isolation

Hi ..................

i am trying to isolate membrane proteins......... while going through the forumi got this protocol Posted By qinglongyanyuedao on 2/12/2009 ..... canb you please say what is the components of this extraction buffer? because i am using RIPA buffer with the following concentartion.... but while doing the blot i am unable to see my membrane bound prortein..... Kindly help me

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    "Times New Roman"">Tris-HCl: 50 mM, pH 7.4
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    "Times New Roman"">NP-40: 1%
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    "Times New Roman"">Na-deoxycholate: 0.25%
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    "Times New Roman"">NaCl: 150 mM
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    "Times New Roman"">EDTA: 1 mM
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    "Times New Roman"">PMSF: 1 mM
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    "Times New Roman"">Aprotinin, leupeptin, pepstatin: 1 µg/ml each
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    "Times New Roman"">Na3VO4: 1 mM
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    "Times New Roman"">NaF: 1 mM

THIS IS THE PROTOCOL POSTED BY qinglongyanyuedao on 2/12/2009

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  1. Extract your cell culture with your extraction buffer (without detergent, which will extract your membrane protein). The original ectraction buffer you listed is for total protein.
  2. Spin down at a lower speed (the speed you used to spin down your cells, I am using 1800g for my cells) to get rid of any un-brooken cells and cell debris
  3. Take supernatant, and spin down at ~60,000g if you have a ultra-speed centrifuge. If not, spin down at the max speed with a table top centrifuge
  4. Take supernatant, which is your soluble protein fraction
  5. Resuspend the pellet with your extraction buffer + detergent (I will suggest 2% instead of 1%). Or simply resuspend the pellet with 1X SDS sample buffer
abcdef
abcdef's picture
hi..................

hi..................

The main thing is i am trying isolate the transmembrane proteins form THP1 cells.............. kindly give me some ideas to get that