I am wondering how one can avoid saturation of loading controls while detecting low abundance protein of interest. I usually load 15-20microgram/lane of nuclear fraction from tissue homogenate to detect my protein of interest but this gives saturation of loading control bands (TBP in my case). I would appreciate your suggestions to overcome this problem as I want to quantify and compare the bands for various samples.
see this it might help http://www.bio-rad.com/webroot/web/pdf/lsr/literature/RP0058.pdf
I did experience the same problem with b-actin Ab (which I used 1:50000 dilution, reused it 10 times). And these were the tricks :
-when you expose the x-ray film to the membrane, put 2-3 films in the cassette for the short time and develop only the very top one (the furthest from the membrane)
-better way is to use fluorescently-tagged 2'Ab and different detection systems (typhoon or Odyssey)
Hope this helps
see this it might help http://www.bio-rad.com/webroot/web/pdf/lsr/literature/RP0058.pdf
I did experience the same problem with b-actin Ab (which I used 1:50000 dilution, reused it 10 times). And these were the tricks :
-when you expose the x-ray film to the membrane, put 2-3 films in the cassette for the short time and develop only the very top one (the furthest from the membrane)
-better way is to use fluorescently-tagged 2'Ab and different detection systems (typhoon or Odyssey)
Hope this helps
If it were me I'd use a dilution series for the control protein - for example maybe 3 points, 0.1ug, 0.01ug, 0.001ug. Set up the gel so that the lowest concentration control is the nearest to your samples so if the higher bands are overexposed they don't bleed over into sample bands. When you've done this once you'll have an idea of the best concentration for future blots.
Hi, I used the second method of the protocol attached.
I prefer to load the same amount of proteins to analyse housekeeping and protein of interest and increase the diluition of primary and secondary antibody, if the housekeeping is saturated. For example if I load 40-50ug of my sammples I dluited actin 1:4000-1:5000 (sigma) and the secondary 1:2000 (anti mouse cell signaling) or more.
Beta-tubulin is also an option. Maybe try to use multiple controls in this case such as a nuclear protein from your sample. Diluting the antibody would work as well just not not have to do it myself. The only other thing is to minimize exposure using the method you are. Typhoon is great to use if there an 2ndary Ab you can use and you have access to a machine. Best wishes in your experiment.
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