pipetting technique for western blot sampling - (Feb/14/2008 )

hi all,
for sampling of western blot, after sonication, when i centrifuge, most of the times i could not see the pellet visually(may be it is there, but too small to be seen).
and when i transfer the supernatant (as cell lysate), sometimes that small pallet comes with the supernatant. the problem might be technical.

i tried leaving a good amount of lysate within the centrifuged tube (i mean from a vol of 200 ul lysate if i transfer some 130 ul) less chances r there that pelette will come with the supernatant. becoz, i don't need all vol of lysate, this pipetting (raw ?) works.

i have the idea that, becoz all through the lysates, protein conc should be the same, so any volume i pick up after centrifuge, should give me the reliable result.

anyone does differ or have some better idea.

i beg pardon, if the post becomes confusing.
thanx.

lilliput

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Spin it at full speed in your centrifuge ~ 15,000x g. Use 10ul pipette attached to a 100ul pipette while removing supernatant so that you donot disturb the pellet.

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thanx scolix to see u appear.
i centrifuge at 12, 000 rpm for 5 min.
i got ur sense. how about the idea of picking up not all the vol of lysate (as i discussed in my 1st post) even if i use a 10 ul pipette tip.

lilliput

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yeh sure, you could leave some of the volume but like you wrote earlier, from 200ul, you pipetteted only 130ul leaving 70ul, I think thats too much to leave behind. A few ul is fine.

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