Iam trying to detect proteins expression (ATM, Rad51, DNA-PKcs ... etc) in my Ph.D. Iam using western Blot right now but Iam looking to use more precise method. Can any one suggest other methods?
ATM is a relatively large protein and therefore quantitative transfer to a blot membrane may bring about problems. This aspect is particularly important when small changes in expression should be determined. In such cases, we used slot-blot analyses to circumvent electrophoresis and 'electrotransfer'.
Another aspect: I don't know the scientific question behind but possibly it may be more relvevant to measure the level of phospho-ATM as this is the active species. We applied slot-blot analyses for pATM and concomitantly determined levels of downstream protein/phosphoprotein components.
Peter
That depends what you are working with. If you are working with Bacteria I have a couple of options for you.
Did you engineer your protein to be expressed from the chromosome with an affinity tag for purification? If you did, purify your protein, and do a BCA assay, which gives fairly accurate protein conc.
If you did not do this, it will be hard to get actual protein concentrations. Another way around this might be to do qPCR and identify the transcription levels of your proteins of interest, which will tell you the relative expression to one another compared to a control, however the strength of the ribosome binding site may change the amount of translation you have, so unless your proteins have the same RBS it may not be entirely accurate.
If suitable antibodies for the protein of interest are available you can use Western-Blotting and subsequent quantitation of band intensities. It is important, however, to apply same protein quantities for electrophoresis to allow comparison between samples. This should be checked by concomitant detection in the Western blot of a protein which is usually not altered in expression by cell treatments. This can be for example beta-actin for which antibodies are available.
Which protein(s) do you intend to quantify?
Perhaps I can give more advice for its quantification.
Best wishes, Peter
Hi Peter, Iam using western Blot to detect ATM protein and my problem is when I use my Inhibitor I see small down regulation and I need sensitive method to precisely quantify this little down regulation!!! I thought at the beginig that WB is the best!!
ATM is a relatively large protein and therefore quantitative transfer to a blot membrane may bring about problems. This aspect is particularly important when small changes in expression should be determined. In such cases, we used slot-blot analyses to circumvent electrophoresis and 'electrotransfer'.
Another aspect: I don't know the scientific question behind but possibly it may be more relvevant to measure the level of phospho-ATM as this is the active species. We applied slot-blot analyses for pATM and concomitantly determined levels of downstream protein/phosphoprotein components.
Peter
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