quantification of a protein in tissue
Posted 09 November 2010 - 02:56 AM
I made western blot of tubulin and my protein in order to quantify the levels of my protein of interest in the brain.
i loaded 50ug of protein extract and i saw bands of my protien on the lanes loaded, later, i did tubulin W.B on the same filter and i obtain strong signal reaching saturation, i could'nt quantify if its saturated! so , i did another W.B on another filter and again tubulin, less stronger signal but, reached saturation in 2 lanes! so, my collegue said that u can't quantify! i tried to do WB of actin in the same filter, it looks good signal but, 2 bands are saturated!!
i feel frustrated to repeat again wb, so, i want to strip this filter and try again actin high dilution and incubation of less time!
do u have any advice ?
Posted 09 November 2010 - 06:07 AM
I am not sure I understand completely what is the exact problem - why is the "saturation" of a band a problem exactly. If you are comparing two very differently expressed proteins from the same sample, you will inevitably get "saturation" if by that you mean that the band is fat and black. WB is by default a semi-quantitative method and if you are working with photographic film it is an estimation method at best. What is in my opinion the best and easiest approach is to do a dilution series of your brain homogenate sample in consecutive lanes (for example in lane 1 you start with 50 ug, in lane 2 you load 25 ug in three 12,5 ug etc...) and then you blot this onto two DIFFERENT membranes and stain 1 membrane with anti-tubulin or anti-actin and the other with anti-(your protein). Then develop both membranes simultaneously and you can compare how many times you had to dilute the sample to obtain the same intensity of bands for tubulin as compared to the undiluted sample intensity for your protein. Some bands will of course be big and fat and some will be almost invisible, but some will be just right. If you know the concentration of tubulin or actin in your sample then divide this by how many times you had to dilute the sample to obtain similar intensity bands and the result is the concentration of your protein. You can even do a dot blot, which is much faster, because you don't have to separate and transfer the proteins. Just apply 1-3 ul of your dilution series directly onto the membrane, let dry, block and stain.
I guess you can do this on the same membrane, but I prefer different membranes so I can adjust the exposure time of the film better.
Hope it helps.
Posted 09 November 2010 - 03:13 PM
in fact, i have already the bands of my protein extract and i'd like to quantify using tubulin in hte same filter obtained from gel where i loaded 50ug, sufficient qantity to see my band of protein of interest!!
but, tubulin bands are stronger ( with white inside the black band, after observation in Image J, which means saturation).
I want to take advantage from the western of my protein of interest, without repeating again all the experiment,
i want just to do tubulin or actin on the filter of 50ug extract.but, it seems not easy to get convenient result to make qantification
im sorry, i hope u understood
Posted 09 November 2010 - 11:12 PM