Advice needed for Western blotting

[Peter Ellis]

Hiya,

I am just starting up a project investigating two novel proteins in 
testis function.  My background is in molecular genetics and transcription 
profiling, so protein work is all pretty new to me.

We have peptide antibodies prepared (or in the works) for both proteins - 
the first step is obviously to characterise the antibodies.  We're using 
peptide antibodies rather than antibodies to recombinant protein because 
there are many related genes, and we want to avoid cross-reactivity.

We have ORFs cloned for each of the genes and their close relatives.  The 
aim is to use in vitro transcription/translation to generate pure 
proteins in order to check the antibody specificity.  Once we know 
that the antibodies do actually detect our proteins of interest (and not 
the relatives), we can move on to other more interesting questions!

So the first plan is Western blots using whole testis extracts to see 
whether we have a band of the expected size in testis.  We'll then do 
further Western blots of testis extract alongside the various in vitro 
translated proteins in order to check AB specificity.  At this point I'm 
looking for advice / sanity checking on how to extract my proteins and 
what sort of gels to use for the Western blots.

On looking around in the literature, I'm finding myself a little 
bewildered by buffer compositions - many papers refer to "standard RIPA 
buffer" or "standard Laemmli sample buffer", and yet there seem to be as 
many different recipes as there are laboratories.  Secondly, it looks like 
the most common buffers simply won't work for my proteins!

Details below for each of the proteins of interest:

*************************************************

Protein 1: H2AL1
----------------

This one is a novel histone.  I've looked at the QIAGEN QProteome Nuclear 
kit, but I'm loath to commit to using that as it doesn't tell you what any 
of the buffers are.  I can't even tell if it's an acid extraction or a 
salt extraction!  Also, it's very expensive for the amount of tissue you 
can process with it.

So I'd prefer to do my own histone extraction if possible.  I believe that 
means I want to prepare purified nuclei and then do an acid extraction. 
Previously I've done nuclear preps when preparing high molecular weight 
gDNA - will the same buffers be appropriate for protein work?  Protocol is 
as follows:

* Homogenise tissue at low speed in lysis buffer [0.05M TrisCl pH 7.5, 1mM 
EDTA, 5mM MgCl2, 50mM NaCl, 5% glycerol, 1% Triton X-100, 1% B-ME], using 
100 mg tissue / ml buffer.

* Centrifuge to spin down nuclei

* Wash in lysis buffer and spin down again


Once I've got the purified nuclei, the histone prep looks admirably 
straightforward.  According to Abcam, you just resuspend the nuclear 
pellet in 0.2M HCl and leave it overnight at 4 degrees C.  Centrifuge to 
spin down debris and keep the supernatant.  However, other protocols seem 
to use different acid concentrations, or H2SO4 instead of HCl, and may 
even include B-ME in the histone extraction buffer.

One protocol I Googled called for you to neutralise the preparation with 
KOH afterwards - is that really called for?  Wouldn't it just drop the 
proteins straight back out of solution again?

Once I've got my histone prep, what buffer should I use for running them 
on an SDS-PAGE gel?  Standard Laemmli sample buffer?  If so, *which* 
standard Laemmli buffer?  Will a normal SDS-PAGE gel work OK, or will 
there be pH issues due to the sample being in pretty much neat acid? 
Generally the lab uses Novex gels in a XCell mini-blot module.  For a 
histone (size ~17 kDa), it'll be quite a high percentage gel.


*************************************************


Protein 1:   mgclh
------------------

Localisation of this protein is unknown, but a closely related gene 
(Gmcl1) is known to be a nuclear lamina protein.  Gmcl1 is RIPA-insoluble, 
so it's likely mgclh will also be RIPA-insoluble.  Protein size is 
expected to be around 55 kDa.

The plan here is to do a standard protein extraction by homogenising 
testis tissue in RIPA + protease inhibitors.  Retain the supernatant (to 
get the RIPA-soluble fraction), and then dissolve the pellet in something 
(to get the RIPA-insoluble fraction).  Question is what to dissolve the 
pellet in.

The original literature on Gmcl1 said they resuspended the RIPA-insoluble 
pellet in "SDS-polyacrylamide gel electrophoresis sample buffer", but 
doesn't give the composition.  I would assume this is 1x Laemmli sample 
buffer.  Does this sound plausible?  If so, what recipe for Laemmli buffer 
would you recommend?  I've found half a dozen different recipes, and the 
only common factor between them is ~60-100mM Tris pH 6.8 and 2% SDS. 
There are variants with and without DTT, with and without B-ME, with 
and without loading dye, using sucrose or glycerol for increased density!

I would imagine I'll need to add B-ME to reduce the proteins before 
running on the gel, but should I add the B-ME at the point I resuspend the 
pellet, or only when I'm about to run an aliquot on a gel?  Will I need to 
add protease inhibitors, or will the Laemmli buffer itself take care of 
that?

*************************************************

Thank you for your time and patience - sorry for asking so many questions, 
most of which will undoubtedly have obvious answers.  Probably several 
mutually incompatible obvious answers, mind you, but that's biology for 
you.  My theory is that it's better to ask silly questions and look daft 
than to forge ahead without asking and cock something up!

Thanks,
Peter Ellis