Alternative Techniques for Protein Detection and Quantification

[gravenewworld]

Does anyone have any experience with assays or techniques that are alternative to a traditional western blot? Why hasn't the WB been made obsolete yet with technology in order to make protein detection and quantification much faster than and more repeatable than running an extremely long WB protocol that can go wrong at almost any step along the way? Are there any alternatives that are much faster but are just as widely accepted as a WB for protein detection?

 

[Ygggdrasil]

How about an ELISA?

 

[gravenewworld]

I looked into ELISA, is it just as good? Doesn't it have problems with false positives?

 

[Monique]

Western blots are not hard.. what is the read-out that you're interested in? An ELISA is not that different from a Western blot, the draw-back of ELISA is that proteins are not separated. You must be sure that the antibodies used are specific and that you're not interested in protein modifications.

 

[gravenewworld]

Western blots are not hard.. what is the read-out that you're interested in? An ELISA is not that different from a Western blot, the draw-back of ELISA is that proteins are not separated. You must be sure that the antibodies used are specific and that you're not interested in protein modifications.

OK, the situation is this:
-I'm just after a single protein to see if it is present in certain types of cell lines. No worries about complexes etc. Just want to see if it is there, and if so, eventually figure out the amount.

-I've never done a WB before, know they are time consuming, and am afraid I'll mess it up and use expensive reagents in the first shot.

-Thought about doing ELISA, but the only antibodies I can find against the protein are polyclonal. Will they still work for the protein then? Polyclonals are less specific right?

-Would the polyclonals work for the WB?

Thanks for the replies thus far.

 

[Kinase]

What is the protein? If you don't mind saying.

 

[Monique]

In that case you could do an immunostaining on your cells: fix the cells and stain. Do you know the specificity of your antibody? You need to be sure that it does not aspecifically stain proteins (take along some control that shouldn't stain).

I do Western blots regularly, it really is not difficult and I've never messed up one. There are pre-made gels that you can use, which saves time. It really is not time consuming, since you can do other stuff in between. Overall the procedure does take 1-2 days to complete.

You can check biocompare.com for an extensive list of antibodies and the assays they've been successfully used in. Also check the literature yourself for a particular antibody for optimized assay conditions. Antibodies that work in immunostaining don't always work in Western blot and vice versa.

 

[saurabhgayali]

You can directly do ms of extracted proteins by top down approach in an MS directly without LC (Like MALDI-TOF) Specify a narrow mass rang covering your protein in the parameters to reduce load on MS.

This approach is superior to western. Western has fault rate upto 10% while MS has fault rtae of 0.1%. Its getting that purity for is hard in case a negative result is expected like in case of organellar purity experiments. But it can be used undoubtedly for protein detection however its just quantitative.

 

[Curious3141]

Does anyone have any experience with assays or techniques that are alternative to a traditional western blot? Why hasn't the WB been made obsolete yet with technology in order to make protein detection and quantification much faster than and more repeatable than running an extremely long WB protocol that can go wrong at almost any step along the way? Are there any alternatives that are much faster but are just as widely accepted as a WB for protein detection?

The most direct "new and improved" analogue to a traditional Western blot is a RIBA, right down to the familiar banding pattern. Purified synthetic antigens are precisely spotted on a strip before antibodies are allowed to react with them. No electrophoresis is involved unlike in WB, so the band spacing can be made completely uniform.

ELISAs and other related assays (IFAs, RIAs) are not really analogous to the WB in the way RIBA is. ELISAs can assay for a specific antigen-antibody reaction, and the specificity can be controlled by using different epitopes vs a single purified epitope, monoclonal vs polyclonal antibody, etc. But when you want to assay the reaction of an unknown antibody mixture to several different epitopes at the same time in a single reaction, then the only choices are a WB or a RIBA.

That answers your WB question, but as for protein detection methods in general, there's a whole lot else to say. Others have already addressed some aspects of this.

 

[gravenewworld]

You can directly do ms of extracted proteins by top down approach in an MS directly without LC (Like MALDI-TOF) Specify a narrow mass rang covering your protein in the parameters to reduce load on MS.

This approach is superior to western. Western has fault rate upto 10% while MS has fault rtae of 0.1%. Its getting that purity for is hard in case a negative result is expected like in case of organellar purity experiments. But it can be used undoubtedly for protein detection however its just quantitative.

We did look into MS, however, sensitivity was a possible issue, especially for proteins with low quantities. Antibody sensitivity is still several orders of magnitude better than routine MS techniques that are used AFAIK. We simply didn't want to have to grow a massive quantity of cells to be able to detect by MS.


Man how I would love to get my hands on an isntrument like this to try it for a trial run:

http://www.proteinsimple.com/simon.html


I'll have to look into RIBA, never heard of it.