IP Protocols and hints

Hi all,

I've recently got my hands on an antibody to a small protein that's part
of an enormous complex (over 100 nm in diameter) and thought I might try
some IP to purify it. I've also got some Protein G-sepharose beads in
the fridge which I could use for IP. Can anyone offer protocols or point
me in the direction of some to help me out? I'll need to do this under
native conditions. I've done very little IP before, so any hints would
be handy.

Cheers,

Bean

I recently succeeded in endogenous co-IP of some erythrocyte
membrane/peripheral proteins. the protocols and recipes in paper below may
provide you with beginning at least. as you know IP is trial and error with
different buffers/ detergents/ etc. However, you mention that you want to
purify?? this protein by IP? IMHO you may end up wasting a lot of time for
unusable amount of protein.
AK

J Biol Chem. 2008 May 23;283(21):14600-9. Epub 2008 Mar 17.
Dematin and Adducin Provide a Novel Link between the Spectrin Cytoskeleton
and Human Erythrocyte Membrane by Directly Interacting with Glucose
Transporter-1.


"Bean Long" <[Only registered users see links. ].edu.au> wrote in message
news:4830d6d8$[Only registered users see links. ].au...

AK wrote:

Many thanks AK. I s'pose 'enrich' would be a better word? The hope is to
do a bit of proteomic work and subunit relative abundance analysis on
it. Enough to run relative pure stuff on a gel would be handy. I had a
little play with the Protein G-sepharsoe I found but ended up with
bucket loads of what I assume is Protein G on my gels. I also think it's
a little too old for successful IP, so I'm getting a new batch!

Cheers,

Bean

On May 23, 2:44*am, Bean Long <[Only registered users see links. ].edu.au> wrote:



More likely it was IgG you saw on your gel. The heavy chains are
between 50-60 kDa and the light chains are 20-something-ish kDa.

Nick

--
Nick Theodorakis
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Nick Theodorakis wrote:

My first thought too Nick, but the same bands showed in my Protein-G
only control... no IgG added!! Any recommendations for good sources of
Protein-A or Protein-G beads for IP??

Cheers,

Bean