Technique to estimate the level of protein expression from media - (Nov/20/2008 )
I am doing an experiment where I have to collect cells after treatment with my drug every 2 hours. To study the expression levels for my gene of interest I do an RT PCR...
Now I have collected the media and would like to measure the level of my protein of interest. I know RIA is one method, but is there any other method that I can do???
Also another general query - My colleague dissolved some DTT in water. to 20mgs of DTT he added 800ul of water. After dissolving it thoroughly, he found out that the volume of water had increased to 1000ul now!! I told him the DTT once dissolved will definitely increase the volume, he said no the molecules in the DTT have occupied some space in the water thats why the level has inceased... Can aybody tell me what the science behind this increase in water level!!!???? Thanks all in advance!!!!!!!!
you are adding mass to the water, although, adding 20mg should not increase the volume by 200 ul.
In order to determine the concentration of your protein of interest, you can do either an ELISA, EIA or RIA. If it is a common protein, look for commercially available kits. If there are no kits already but there are readily accessible antibodies and a pure form of the protein, you can easily set up an ELISA methodology. The nice thing about ELISA and EIA - no radioactivity.
Hope this helps,