What's the difference between ELISA and Western Blot in terms of quantifying membrane proteins?
The biggest difference is that you can easily verify that your antibody only reacts with a single protein (or at least molecular weight target) by Western, but all targets in your ELISA will be captured (possibly at different affinities, so subject to time and temp as variables). ELISA reports a single, total quantitative measure and WB a much broader array of data with about 1-2 orders of magnitude lower total signal range.
I am not sure, if I understand your question, but these are two completely different methods. From my experience I would say its easier to quantify ELISA, but you need a proper standard here (your protein in known concentrations). If this is not available, you have to use western blot and do an estimation on the bands.
If possible, I would choose ELISA over western blot, since its more precise and allows absolute quantification.
Hi. I agree with Christian...Those are totally different methodologies. With ELISA you can do an absolute quantification, keeping in mind that you need to have a standard curve made of your protein of interest. In WB you do a relative quantification, you do not need a std, but you need an housekeepin protein to normalize the intensity of the band of your POI. Hope this helps, ask if you need more
Both techniques are used to detect the soluble protein released from the cells or protien that is expressed by cells against standard curve/ positive control like others mentioned. Both of them are immunoassays and can be used in detection of proteins. ELISA gives more quantitative measurement of the protein released or present in the cells.
ELISA allows you to get quantification (e.g. ng/ml) of your protein . Therefore you will have an absolute quantification of the protein by interpolating your values to a standard curve made with known quantities of your protein of interest. WB can give you information about relative amount of protein normalized to an houskeeping protein (e.g. Actin, GAPDH etc). I would say ELISA is much sensitive than WB and of course gives you absolute quantification. If you need extreme sensitiveness you can try ELISpot assay.
I agree with Christian as well. However, depending on the ELISA, you can get artifactually high results if there is any cross-reactivity with other cellular proteins. Generally a sandwich ELISA with two different monoclonals is reliable. In my experience the only use for Westerns is to see 1)How specific your Ab is, and 2)get an overall feel for what is going on with your protein (eg. it goes up a lot, it is phosphorylated, etc.). For absolute quantitation I would not trust a western, there are just too many variables that effect the signal.
The biggest difference is that you can easily verify that your antibody only reacts with a single protein (or at least molecular weight target) by Western, but all targets in your ELISA will be captured (possibly at different affinities, so subject to time and temp as variables). ELISA reports a single, total quantitative measure and WB a much broader array of data with about 1-2 orders of magnitude lower total signal range.
In terms of cost western blot is quite complex and is expensive and time consuming as well as difficult to automate. ELISA is simpler and doesn't involve use of SDS-PAGE and nitrocellulose sheets transfer but is performed on 96-hole plates. ELISA can detect native proteins in serum given the fact that it does not involve electrophoresis. elisa is highly quantitative unlike western which is more qualitative and vaguely quantitative. elisa is prone to false positives due to cross-reactivity. western takes care of crossreactivity by first separating the different proteins in terms of size through electrophoresis.
I ran a western with 2 sets of samples + control. Non-reduced vs Reduced.
When I try to quantify the samples (reduced vs non-reduced) based on the control (reduced and non-reduced). It was very different !!! The reduced had 2 clear and sharp bands vs the one fainted band of non-reduced. I also wanted to correlate these quantifications to ELISA but it gave me a headache !!! Could you kindly give me some answer ???
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