Cell Lysate Preparation (Native)

( www.abnova.com ) - Here we display you the protocol on how to prepare cell lysate. This includes 3 steps: First, cells are harvested. Then, cells are lysed in lysis buffer. After ultra speed centrifuge, cell lysate is prepared as ready-to-use, native lysate in modified RIPA Buffer. More videos at Abnova www.abnova.com

[@8scatterbrain8]

you detach cells with PBS???

[@MattbyNature]

+8scatterbrain8 Yes. Hek is not that adherent - I do it all the time with them and it works fine. For splitting I wouldn't do it though because you can't separate them from each other without trypsin.

[@8scatterbrain8]

+Lal3l oh ok, I know what you mean, I worked with those kind of cells too..but maybe you should've said that in the video because it's not routine (even though everybody knows you have to use trypsin to detach cells!(: )

[@HaujiunLian]

RIPA is a denaturing lysis buffer

[@essenzadeisogni]

Three criticism:
By adding PBS and pipetting not all adherent cells detach from the dish.
The speed in rpm doesn't mean anything for people using different centrifuges.
I might be wrong but as far as I know RIPA is a denaturing lysis buffer so what's the meaning of "native"?

[@parkerwebster2571]

Three points of support: (1) Stating facts about adherent cell dissociation probabilities is not criticism, until the opinion jury reaches an opinion. (2) (2) Speed in RPM doesn't mean somethings to (possibly many) people, with or without a centrifuge; The speed limit of a road can be discovered by (not) reading the signs. (3) RIPA denatures things. The author utilized 'Modified' RIPA Buffer. www.novusbio.com/support/general-support/buffers.html; Good question: What IS the meaning of "Ney-tiv"? ... What's my meaning?... and Who is What for Why?.... but how?!.... ahh, such as whenner.

[@AHSinNYC]

Definitely do not use RIPA buffer for non-denaturing (native) PAGE! This video is wrong!

[@parkerwebster2571]

it's modified ripa buffer. water's a modified RIPA buffer, right?

[@ChangeOrBeCrushed]

this video is wrong on so many levels. the experimenter also has idiotic lab skills.

[@hangcai]

can you tell me more about the lab skills? I just started working in a lab. Thank you!