Login

Join for Free!
116861 members


Is an ELISA plate really specific for protein adsorption?

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Is an ELISA plate really specific for protein adsorption?

Postby hanhan2008 » Tue Dec 04, 2007 3:39 pm

Not lipids, DNAs, etc. ? What about lipoproteins and glycoproteins? Could anybody provide some basis for this specific interactions of protein molecules w/ surface groups at the bottom of the ELISA plate wells? Or links to some orginal research papers? Thanks.
hanhan2008
Garter
Garter
 
Posts: 26
Joined: Wed Sep 05, 2007 5:41 pm

Postby woolleyy » Tue Dec 04, 2007 4:13 pm

A sandwich ELISA has three components, two antibodies and a molecule that is recognised by both of the antibodies. The molecule is most often a protein, but theoretically could be any molecule that has an antibody that will specifically recognise it. I know that antibodies can be made for many non-protein molecules, some extremely small. Does anyone know about their application in ELISAs?
The ELISA technique is not only used to identify whether a protein or other molecule is present though. It is possible to test for the presence of specific antibodies also.
Hope that helps,
woolley
User avatar
woolleyy
Death Adder
Death Adder
 
Posts: 77
Joined: Mon Nov 19, 2007 2:01 pm
Location: Aberdeen, Scotland

Postby hanhan2008 » Wed Dec 05, 2007 2:55 pm

Thank you. I guess ELISA plates are typically pre-coated with a certain antibody. I forgot to mention that I intend to ask those questions on non-typical plates: those ELISA plates with no antibody pre-attached.
I'm trying ELISA method for detecting protein carbonylation (a type of protein oxidation due to cellular oxidative stress). Carbonyl group (the antigen) can be found in almost all proteins, though some proteins may tend to be more carbonylated than others. In this case, one needs to attach antigen-containing proteins as a first step before antibody detection .That's why I doubt if the first step is really specific (no lipids and others are attached) and I want to know more chemistry for this specificity.
hanhan2008
Garter
Garter
 
Posts: 26
Joined: Wed Sep 05, 2007 5:41 pm


Postby andyde » Thu Dec 06, 2007 1:19 pm

Every entity (each protein species, lipid molecules...) has it specific affinity towards the polystyrene surface (also depending on the PS-type and pre-treatment). So, if you take a crude extract, you will never bind all molecules and never in the same proportions as they are present in solution. But if you charaterize your system thoroughly it is a an easy system, anyway.
You have to normalize your results to proteins for which you know that their content never changes (house-keeping genes, structural proteins, histones, ribsomal proteins or even DNA).
Hence you get the relative content of your carbonylated species vs non-carbonylated in relation to the reference protein.

What is the organism you are working with?
andyde
Garter
Garter
 
Posts: 2
Joined: Thu Dec 06, 2007 12:58 pm

Postby hanhan2008 » Fri Dec 07, 2007 2:48 am

Thanks a lot! I'm working with a rat epithelial Type I-like cell line, called R3/1. If this works, I may extend it to human microvascular endothelial cell line, and lung tissue (and even other tissues) of rat.
hanhan2008
Garter
Garter
 
Posts: 26
Joined: Wed Sep 05, 2007 5:41 pm

Postby woolleyy » Fri Dec 07, 2007 10:14 am

When you do an ELISA (or at least, how I did an ELISA, the one and only time I've done one) was using a normal 96-well plate, with nothing pre-attached to the bottom of the plate. Each of the components of the sandwich are added during the experiment. So the antibody (or crude extract, depending on what you are looking for) is added first, and the proteins will bind to the bottom of the plate. This is washed with detergent (which helps to fill the areas of plastic with no protein yet bound, preventing non-specific attachment in the second part), and then has the crude extract (or specific antigen, again depends what you're looking for) added to the wells. Finally, an antibody(for the antigen)/enzyme conjugate is added, to allow recognition of the antigen bound to the antibody already there. The substrate for the enzyme part of the conjugate protein is then added. The substrate chosen must have some method of analysis of the reaction that occurs, for example a colour change.

Okay, you probably know most of that, but I started writing and just couldn't stop! Hope there's something in there that helps,
woolley
User avatar
woolleyy
Death Adder
Death Adder
 
Posts: 77
Joined: Mon Nov 19, 2007 2:01 pm
Location: Aberdeen, Scotland

Postby woolleyy » Fri Dec 07, 2007 10:19 am

Oh, yeah, was wondering about what you're trying to analyse here? Are you using the ELISA to look for the presence of carbonylated species? Or are you trying to look at the extent of carbonylation on each protein? I think the first method will be easy to do with an ELISA, but the second may be pretty tricky... Let me know, thanks,
woolley
User avatar
woolleyy
Death Adder
Death Adder
 
Posts: 77
Joined: Mon Nov 19, 2007 2:01 pm
Location: Aberdeen, Scotland

Postby hanhan2008 » Sat Dec 08, 2007 3:46 am

Thanks. Your long writing is helpful. One point is about detergent that is used as blocking reagent. I tried Tween 80 at a concentration (suggested by a paper, I forget the concentration, may be 0.1%?). However, it turned out to be less effective than proteins as blocking staff, like reduced BSA (milk is another one, which I didn't try).

I'm trying to quantify the carbonylation in cell lysate in general. I may pursue the same in specific proteins (via other methods) only if this general thing works.

Unfortunately, at this time, though both negative standards and positive standards worked, the cell lysate samples have not. Both standards were prepared using vigorous chemical reactions, while the samples were treated with the exposure of interest, nanoparticles at certain concentrations in my case.
Again, I appreciate the valuable inputs from you guys.
hanhan2008
Garter
Garter
 
Posts: 26
Joined: Wed Sep 05, 2007 5:41 pm


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 2 guests

cron