WB for Membrane Protein - Not able to see membrane protein on western (Aug/03/2005 )
My name is Adithi and I am writing from India. I have some queries regarding the extaction of membrane proteins and I am hoping someone can help me.
I am tyring to extract cell adhesion molecules (N-Cad, E-Cad, tetraspanins etc) from retinoblastoma tumor tissue. We use a lysis buffer with prot inhibitor cocktail, EDTA, Na deoxycholate, NaCl and Triton X 100. We homogenise (on ice) and pass our lysate through 21-gauge needle and then spin it at 5,000 rpm 1-2 times, till clear. We then do a protein estimation by Lowry Method and standard SDS-PAGE (10%) and then transfer onto nitrocellulose membrane at 100v for 1.5 hr. Then we do standard blocking with 5% NFDM (1 hr), primary ab (monoclonal) for overnight, secondary antibody for 2 hours and detect using Amersham’s ECL kit. I am not able to get any signal for these molecules, but am able to get good Beta-Actin bands.
maybe you could try a more sensitive detection system, as the Super signal west femto substrate (or something like this) from Pierce. It is very sensitive, may be you will be able to detect your protein...
Thanks for the suggestion, Nat. Will try and get back to you on how things work with a more sensitive detection system. My worry was that even the positive control (whole cell lysates from Santa Cruz) were not showing up. Maybe this will help.
Is there anything else I could do regarding the lysis preparation? I guess Triton X-100 would be breaking down the cell membrane but do you think sonicating the sample to further lyse it would help?
Antibodies are from what company, My personal experience is, I was also isolating membrane proteins and antibodies were from santacruz which are horrible... simply horrible.
If the antibodies are not working then positive controls are of no use.
I used MDA-MB-231 cell line which overexpress alpha6 and beta 4 integrins however the antibodies were too bad to recognise the protein (in IF, WB, IHC all)
You mentioned Beta actin bands u are able to visualise, indicate that the system is working fine with other antibodies. However what I use is a strong detergent (SDS). I use SDS lysis buffer (20mM Tris HCl pH 7.2, EGTA 5mM, EDTA 5mM, SDS 0.4% and protease inhibitor cocktail from calbiochem) which works fine with all my blots (7 different antibodies).
Thanks for the suggestions! I have had mixed responses regarding santa cruz antibodies, so i will try to see if i can get from other companies.
Which other companies can i look for positive controls from? any suggestions on that?
Thanks once again
You are SURE you have the protein you are looking for? I've been working with membrane proteins too and I've found that I don't always get sufficient quantity of the one I'm looking for, although the Lowry assay tells me that there is plenty of protein in the sample.
I tried upping the quantity of cells I was using and it seems to have worked slightly. However Western blotting just dosn't seem to work properly for anyone in our lab and I've had three totally different results for one sample!
Have you tried an ELISA assay?
Hi! Thanks for your suggestions!!
Yes, I am quite sure that these proteins are present in the samples, as i have confirmed their presence by immunohistochemistry using Santa Cruz antibodies (which, i hear are not very reliable!).
In any case, it is known that many membrane proteins form multimolecular complexes and my worry was that whether Triton -100 and Sodium deoxycholate were enough the disrupt them. Someone suggested adding SDS and EGTA, which I will be trying soon. Any other suggestions?
Would ELISA be as sensitive as WB???
I THINKELISAS are about as sensitive as Westerns (maybe somone who has tried them will be able to confirm this). I'm trying it out of shear desperation as I've been doing Westerns for 9 months straight and not managed to achieve a duplicatable result yet!
Has the use of arcane voodoo rituals been disproven as an aid to getting Western blots to work properly?
Oh well...yes maybe it would be useful to find out if they are as sensitive as westerns!
I know how frustrating westerns can be- i have been trying for longer than you have! why don't you give a detailed protocol and we can see how/where things can be changed? until then...
i was wondering about the specificity of ur Abs
u have mentioned that u used them for immunohistochemistry which mean u detected native proteins with ur Ab
In SDS page there is no native protein. So can it be that ur Ab are specific to sttructural epitopes.
Try a native PAGE to confirm
By the way i am from india too but studing germany right now