Post subject: What's the Place for Acetone powder?
I'm a fan of this site, and I'm a technical kind of guy.
Conundrum: I need immuno-localization of novel protein in mouse tissue;
Reagents: I have mouse (knockout and wildtype) tissues on slides,
KO & WT mice in the colony, and a polyclonal rabbit antiserum against the novel protein.
Question1: Can I take a tissue from a Knockout mouse, make 'acetone powder', preabsorb/adsorb the antiserum, and use the resulting supernatent as a specific marker for the novel protein?
Question2: Does the 'acetone powder' need to be prepared from the same
KO tissue that the immunolocalization in the WT will happen (ie...Liver
for liver, and brain for brain; or liver for brain, and brain for liver is OK)?
Thanks. Any help with the basic (immuno) chemistry of 'acetone powder'
in a practical sense will be very useful.
Thu Sep 23, 2010 12:32 am
Joined: Apr 22, 2009
Haven't used the method, but your success might depend on the ko phenotype. If your polyclonal prep binds another protein that is normally present in the wt tissue but is absent from the ko tissue (expression suppressed or the cell type expressing it is missing), you won't get rid of that activity using a ko powder.
Is affinity purification that much more difficult? Carl?
Thu Sep 23, 2010 9:14 pm
Joined: Oct 11, 2004
Post subject: Hard work....
1. Do you know what the distribution of your protein is, in tissues/cells?
2. Have you immunostained these tissues (ko and wt) with your Ab?
3. What type of sections have you got? Personally, I wopuld ensure that I have fresh snaap-frozen as well as Pwax sections. Sure, perfused fixed cryosections and vibratome tissues would also be v.useful, for novel protein screening as just one method of tissue prep. may give you false negative results.
Have yopu transfected your protein into cells? Then probed these cells with your new Ab?
Imho, forget about any form of purification for the moment....just get an immunosignal signal that is appropriate for your protein in wt tissue/ transfected cells with your new Ab.
xf is correct regarding the KO status....you may have a conditional/partial KO ( your Ab is directed against a part of the protein that is still being made).
Have you ELISA'd and also WBlotted at all with your Ab?
If you get a signal in your wt tissues that is appropriate, see if the same signal is present in your KO. Then, absorb yopur Ab with the immunising peptide and see if your signal is gone.
THEN do as xf suggests....peptide purify and compare results.
Sure, acetone powders from wt and ko matched tissues will also be a v.good control.
VWR or Sigma sell them.
Let us know some more teckie details, please. Such as your immunomethod/tissue preps.
SOrry that this is a muddled answer...so many variables until we know more details.
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