If you load the equal amount of total proteins samples in the gel and then do western blotting, you can quantify particular band by using densitometry.

If you load the equal amount of total proteins samples in the gel and then do western blotting, you can quantify particular band by using densitometry.

Actually you cannot. You MAYBE able to measure it reasonably . But the exact stoichiometry of the Comassie stain binding to protein is not linear. Your best bet is to do a Western Blot and then do a densitometry. But that has its own set of problems. If you have an access to LiCor like system, it may solve all your problems.

Thanks guys. I was thinking towards the same direction because the protein/RNA were extracted from the same quantity of tissue (50 mg), all of which were subjected to the same experimental conditions. So, I will simply run the SDS-PAGE with fingers crossed, and load the same gel with equal amount of ALL samples and then blot. If I get some differences during the densitometry, I could then relate to my experimental treatments compared to the control.

I agree with Jennifer. You won't know if the DTT treatment affected the epitope(s) that your antibodies bind to until you actually try it. You may be lucky - even with monoclonals.

I would try the ELISA. DTT usually protects from denaturation, and only causes it if there is a crucial disulfide bridge in your protein (as in some small secretory proteins). If DTT is still present in the protein extract and you're worried about it affecting the primary antibody, you can inactivate it by adding glutathione (oxidized) to the protein extract.

Best option for you will to go for SDS-Page followed by in gel digestion and then you Can go for liquid chromatography or LC-MS for more accuracy. You can do this for all of your samples from different treatment and then you can analyse LC data of all 4 samples to get quantitative protein concentration with very high accuracy. Only limitation could be availability of HPLC.

Say, am I motivated to try the ELISA!

now to specifics;

Jennifer and George, my primary are polyclonals, so I will give it a try

Norbert, pls may I know how much of the glutathione (what molaity) I need to add?

Rohit, sounds like a good idea but seem a longer route to solve my problems, considering my sample size; even though we do have all you mentioned.

Anuj, for the ELISA the primary was pre-coated to the plate commercially so I do not have it, unfortunately.

Thanks everyone, will give the ELISA a try and report back if I notice any complications that require brainstorming.

Actually SDS PAGE alone does not give you the specific protein u r looking at. You have to do a western blot and it will give you the levels of your protein of interest specifically

i agree with Mallu that u should go to western blotting and equalies for the amount of protein between samples with one of the house keeping gene protein (GAPDH or B-actin) and then measure you desired protein band

it's easy to run SDS and do westernblot and measure witht the help of densitomettry

Reaction of DTT with oxidized glutathione I believe is 1 to 1, so if you add oxidized glutathione to the same molar concentration as DTT it should work. Maybe a little more to drive the reaction, but not too much or you might cause some protein aggregation. Make sure you use the oxidized, not the reduced glutathione.