With a large number of samples and when you want to have a high precision, I would always choose ELISA. Its easier to handle larger sample numbers there in 96 well plates and you can easily quantitize the read-out with dilution series of a standard. Western blot bands can be quantified based on a known standard, but I would call this semi-qunatitative.

With a large number of samples and when you want to have a high precision, I would always choose ELISA. Its easier to handle larger sample numbers there in 96 well plates and you can easily quantitize the read-out with dilution series of a standard. Western blot bands can be quantified based on a known standard, but I would call this semi-qunatitative.

I agree with Christain, WB with 100 samples will be too time and efforts consuming. ELIZA is one of the possible deciesion to simplefied anaylsis. However, you can alos use westren dot (instde of blot). In this case you can save time and reagents. http://westernblot.org/dot-blot/

Good luck!

Both methods are only as good as the antibodies that they employ. Antibodies are not commodities and many companies sell antibodies that simply will not detect endogenous levels of the intended protein. Test both assays with a positive and negative control (cells with Glut1 protein and the same cells without Glut1 protein) to see if the assays even work. Most of these assays will only have been validated with a recombinant protein. This says nothing about whether the signal you get from your cells actually represents Glut1 protein.

Thanks to all, for sharing your knowledge. Dear Mark Livingstone, as you told about negative control, can i take any other cell population, which does not express GLUT 1. Because, normal human RBCs are highly expressed with GLUT 1.

Hi, Annudarai, as a negative control you can use pre-immuno blocking solution. In this case you will have an information how specific is your antibody. Moreover, I would suggest to run WB at least for few samples. In this case you can validate the protein size. Good luck!

The ideal negative control would be precisely the same cells with the protein knocked out or knocked down. Clearly this would be quite difficult for someone working with 100 human blood samples. If you were to do a Western blot and have HeLa cells +/- siRNA knockdown run as positive and negative controls and see a band at the same molecular weight in your samples, this would be good evidence that the antibody is recognizing Glut1 in your samples. For an ELISA, a positive and negative control might work great for a different cell line, but your cells might express some proteins that the ELISA cross-reacts with.

All of the talk about "reproducibility" these days mostly indicates that your responsibility as a scientist is to just indicate which antibody or ELISA you used so that people can reproduce your results. It's not really your job to be certain that the signal you are detecting is really Glut1.

ELISA is a method of choice (given standardized, sensitive, monoclonal antibody and detection system) to detect NATIVE or DENATURED ANTIGENS and to MEASURE ANTIGEN QUANTITY against control. In general, ELISA can be much more sensitive than the equivalent Western blot.

Western blot is a method of choice to DETECT DENATURED ANTIGEN and to confirm its size. WB is NOT suitable for QUANTIFICATION of antigen, one can only roughly estimate the amount of a target protein as more or less than the control and how many fold.

I would be very sceptical about using WB for quantitative analysis of expression: As already stated at best it is semi-quantitative. There is a very narrow window of linearity in response using HRP secondary and so loading has to be very precise. Usually researchers use a house keeping gene (i.e Tublin) as a loading control to compare with as a ratio.

Elisa is generally more sensitive but it does depend on the antibodies used. If you are using a commercial kit that is specifically designed for detecting GLUT1 in human blood then I would hope that the kit has been validated for this and that cross reactivity/background has been determined and should have internal controls.

The method of choice is determined by what you need: If you need quantitative analysis then Elisa is the way I would go.

If your kit has not been validated then you will have to do this using suitable controls i.e cells without Glut1 as stated.

good luck

It depends on what you are trying to do. Fundamentally, ELISA & WB both detect protein, but ELISA detects conformational epitopes & WB generally detects linear epitopes. The same antibodies generally do not work for both procedures. I agree with everyone here: WB on 100 samples would be very time intensive and quantitating would be challenging. ELISA offers you a easy format (2 96 well plates and all your samples would be quantitated, with controls & standard curves) and it is more sensitive & quantitative than WB.

It is essential to understand certain basic principles and mechanisms of immunoassays and antigen‐antibody reactions. In principle, immunoblot and ELISA are identical systems, differing mainly in (i) the nature of solid phase (i.e., polystyrene [PS] in ELISA and nitrocellulose membrane [NC] in immunoblot assays), and (ii) the reading of the reaction or end result (i.e., by measurement of optical density [OD] in ELISA and by visual interpretation in immunoblot assays). In terms of sensitivity, the detection limit of the immunoblot assay is considerably higher because of firmer binding of proteins onto NC than onto PS and because of non-leaching of proteins during washing steps [Tijssen P. Practice and theory of enzyme immunoassays: laboratory techniques in biochemistry and molecular biology. Amsterdam: Elsevier Science, 1985.]. Although the specificity of any immunoassay would depend on the type of probe used in the system, if defined antigens are used either in ELISA or in immunoblot tests, both should yield identical results—at least qualitatively, if not quantitatively. The advantage of the immunoblot test is that one is able to analyze and characterize individual antigenic responses, whereas in ELISA one is able to detect only immune response when defined antigens are used in the system. However, in detection of antigens, an ELISA can result in a false negative, whereas an immunoblot test can be positive. This is because of complete masking of antigens by specific antibodies in immune complexes that would be unavailable for detection by ELISA. In immunoblot tests, immune complexes are made to dissociate before they are resolved on the polyacrylamide gel and subsequently transferred onto NC, and, thus, antigens are detected.

Hope now it's made clear in choosing the immune assay system.