6 replies to this topic

[StefyDE]

Hello to everybody!
I have some trouble with the western blot!
I just got bands of high molecular weight protein and nothing about low molecular weight..
But i was looking for histones modifications,
I did the whole protein precipitation and not the acid one, can it be because of it, so i lost all the histones? Or it doesnt matter that much??
Any suggestion on tranfer tecnique tips?!
Thank you all!
Stefania.

[casandra]

Hi Stefania and welcome to the forum. It would be better if you provide more details about your WB expts- sample preparation (and how much protein did you load etc). And what's whole protein precipitation? You spun the cells down and directly lysed in SDS loading buffer or ?, lysis protocol (did you add inhibitors etc), running (eg gel percentage, voltage, and for how long) and transfer conditions (these are small proteins so they could have blown through the membrane etc.) You only saw high MW proteins so do you have positive control for your antibody eg Na butyrate or colcemid-treated HeLa cells (for phosphorylation or acetylation)? You definitely enrich for histones if you do acid extraction and then run in acid urea gels but you can always try first the standard SDS PAGE protocol.

[StefyDE]

Hello!! Thanks for the reply! I first did the extraction with the lysis buffer ( with protease inhibitors), and i did the SDS page  i loaded a very little ammount of protein, but it cames from the quantitation protocol. I runed at 10V overnight..maye i have lost my protein? Or the didnt transfer?!? Thank you so much!!

[mdfenko]

have you run the gel and stained (not transferred) to confirm that your proteins were in your extract?

if so, and the result was positive, what were your transfer conditions (membrane pore size, time, voltage, tank or semi-dry, etc)?

[StefyDE]

Actually i didt stained, i just did the ponceaux red after the tranfer..the membrane was 15% acrylammide, 10V overnight!

[casandra]

StefyDE, on 01 October 2012 - 07:05 AM, said:

Actually i didt stained, i just did the ponceaux red after the tranfer..the membrane was 15% acrylammide, 10V overnight!

You run 10v overnight on a minigel? You're probing for which protein- a methylated H3 ~ less than 18 Kda? Which protocol are you following? And did you see your low MW standard (10 or 15 kDa) after the run? Try running at 100 V for 1.5 to 2 hours. And how much protein did you load- if you're using whole cell lysates, you may have to load more bec histones (esp the modified ones) are not exactly abundant...and do you have positive controls? Please post more details-  eg transfer, immunoblotting protocols so that other members here can help you better.

[mdfenko]

in addition to what casandra said, you should also run a gel to stain (not transfer) to ensure that your preparation contains the protein of interest (or, at least, a protein that migrates to where you expect to see your protein).

once you are sure that your page conditions are appropriate then you can run a gel for transfer and immunostain.