Western vs. ELISA - (Feb/20/2008 )
Naive question: could ELISA replace western in every case? What are the advantages and disadvantages of western blot compared to ELISA? Thanks!
ELISA works on native protein, while Western works on denatured proteins from SDS_PAGE. ELISA cannot give any information on the purity of the protein. At least with SDS-PAGE and Westerns, you can see how pure your prep is. ELISA can sometimes underestimate how much protein you have, because it only looks at correctly folded protein: this is in some ways better than Westerns, which can't discriminate between correctly and incorrectly folded protein.
Westerns, however, can make nicer images than ELISA data. Also, you can see if you have degradation products on Western.
If you first immuno-percipitate your desired protein and then western it, you can achieve the same as ELISA.
The O.D. you get in ELISA can be because of contaminating protein which cannot be determined unless you do western
hi this is an interesting question,
i will try to put the strenghs of ELISA which fully or partly cant be applicable with WB.
1-in ELISA one can do competion studies (peptides etc) and interaction studies (proteins etc), but not in WB.
2-ELISA takes less time than WB and consumes less antibody.
3- cell based ELISA is important for receptor recognition, but not WB
4-ELISPOT is used in cytokine production, but not with WB (WB-SPOT??? )
5- Immuno PCR highly sensitive technique in ELISA format but not in WB.
6- epitope mapping can be performed with ELISA, not with WB.
7-ELISA is amazingly useful in screening techniques (eg-phage display technology, Monoclonal and polyclonal antibody production), where WB cant be used. moreover if one have a pure recombinant protein it does not necessaly need western blot for confirmation.
8-ELISA can determine the temparature sensitivity of a given protein (denautured or notdenatured), but not WB.
-the specificity of antibody is crucial for both applications
-for WB one must use Ab that recognizes the linear epitope, confirmatioal Abs are most of the time useless.
-lack of capturing system in WB makes it less sensitive over ELISA.
advantages of WB have already been mentioned by other members.
, i agree with leelaram as the cross reactivity of any antibody cant be visualized in ELISA but with WB.
welcome for comments on any of the points i was speaking about..
regards to everyone
western bots allow you too look for modifications of proteins that may not show up in Elisa (i.e. if the protein is still functional, same binding site for an antibody etc).
So you can look at acetylation, methylation, sumoylation, phosphorylation, ubiquitination etc etc.
Also the specificity of western's allow greater statistical significance for smaller variations of pretein amount then ELISAs - where the variability seems to be a little more.
It's damn impossible to western 96 samples at a time though