I want to estimate GLUT4 antibody in my animal models. According to a number of publications western blot is the primary method of estimating GLUT4. But is ELISA an option? Why is Western Blot the preferred method? If I decide upon ELISA how credible will my results be?
But the anti-GLUT4 antibody is plated into my ELISA plate as I'm doing it by kit method. How will I be able to do Western Blot? DO you think in this case its better to do the ELISA without kit method?
ELISA technique is a quick method to quantify the antibody raised against GLUT 4. you can use a manual ELISA method which you actually looking at the antibody titre against GLUT 4.
Hello Sriharsha! I prefer to perform first Western blot to check if there is any protein of interest and whether it is not modified in some ways. When you buy an anti-GLUT 4 antibody already conjugated with secondary antibody the whole procedure take not more than 24h. Also Western blot method is more sensitive as you don't need a lot of protein. You can reveal the blot using Femto Luminol which in highly sensitive. As for ELISA I would set my own antibody concentration and not count on the manufacturer recommendations. Good luck!
I agree with Rick on this one. Additionally, if you are new to western blotting, it can be a nightmare to get right (just speaking from personal experience). ELISA is usually quicker, straigh forward, quantitative, more sensitive and more specific for your target (keeping in mind that WB uses one specific antibody against the target whereas ELISAs usually use two specific antibodies in a sandwich format). That said, Aleksandra seems to have done some work on this before so well worth listening to that too.
Dear Indumati Sharma,
selection of western blot or ELISA depends on the purpose of your study. what you want to do, quantify GLUT 4 or detect it or compare it in different tissue? if you want to see the increase in expression of this transporter following some treatment or compare expression of these transporters in different types of cells. the two methods have their own advantages and disadvantages. So it depends on what you want to do rather than what is available with you. I request you to explain your experiment.
Just to confirm are you wanting to quantify GLUT4 or anti-GLUT4 antibody? Generally I have used ELISA for proteins that are secreted so use the culture media as the sample and westerns for protein from cell lysates. Its always easier to justify he method if someone else has done it. i.e. if people have used both westerns and ELISA for GLUT4 then it doesn't matter so much; as you say if they are predominantly western blots then try and go with that. Just double check to see if the studies are measuring GLUT4 or anti-GLUT4 antibody :) I have never quantified an antibody but assume in principle that an ELISA is the method to use.
western blot is used to determine how much of a specific protein is present in your samples and not to estimate the amount of antibody. I've never heard of using a Western or an ELISA to determine the amount of antibody present.
If I understand you right, you already use an ELISA kit. In most cases a commercial kit consists of two antibodies against the antigene, one to bind it to the ELISA plate and a second to detect it which is coupled to an enzyme for detection (mostly peroxidase). If you really want to do a Western Blot with this kit you can use the second antibody for the detection but it is possible that you will get a lot of background.
I'm agree with Patricia, WB is only used to determine how much of a specific protein precipitated for example is present in your samples.
The prerequisite for an ELISA is a highly purified antigen. If you have this, you can run a direct ELISA (solid phase: antigen GULT4 // sample with antibody // anti-species-enzyme labeled // substrate reaction // stop // measurement).
If you have no purified antigen, you can use an indirect ELISA version with one incubation step more: (solid phase: anti-GLUT4-antibody // GULT4 crude antigen mixture //sample with antibody // anti-species-enzyme labeled // substrate reaction // stop // measurement). This requires an specific anti-GULT4 antibody either mono- or polyclonal. This antibody on the solid phase will catch the antigen only in the next incubation step. All other steps are comparable. You have to take care only, that your anti-species-antibody (labeled) is not cross reactive with the solid phase antibody. Anyhow, you need just on control well more on your plate.
If you have once established your ELISA, you will be cheaper and faster than any Western blot. You will have another advantage: you can run more sample per experiment and you can introduce a calibration curve by using an antiserum/antibody and defining a given dilution as standard with e.i. 100 AU/mL.
See figure 4 in the attached paper (indirect assay).
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