i have some simple questions on protein assays. I have been using Biorads DC protein assay kit and a Genova spectrophotometer.
i think i've been getting good results with the Genova supplied cuvettes which have a narrowing towards the bottom which allows 1mL to fit in that column. My first question is since I am running out of these sample cuvettes I have some old polystyrene cuvettes that don't have this narrow column so when I put my 1mL sample in, it only occupies a fifth of the height of the cuvette or maybe half the height of the Genova cuvette. At first I thought it shouldn't matter but each of my spec readings were either negative or very low even though there's clearly color in the tubes. Obviously i need more volume in these cuvettes but other than consuming more of my protein samples for these is it possible to just dilute the color reacted final samples with like another one or two mL of distilled water to obtain the desired cuvette height?
This leads me to the second question. I've always wondered why the manual for this kit says I should use 100ul of my sample or 5ul if it's a microplate assay. I have been just scaling everything down instead to save on what little protein I get from my extractions. That should be ok as long as all the proportions are correct right?
Also related is I found a quartz cuvette and when i used it i think my results were good except all the ODs were just approximately twice as high. I asked the postdoc about the cuvette and he said it was a UV only cuvette but he wasn't sure if it would make a difference. Can I trust this quartz cuvette or should i stick with the polystyrenes. I guess I wanted to use this because we are down to our last three of the Genova cuvettes.
Lastly, I been using BSA 2mg/ml as my standard and I am not sure how to create my curve. I read I should have it at least expand two logs but the manual says to keep it between 0.2mg/ml and 1.5mg/ml. I think I read that the linear range of BSA is higher than IgG but I think some documents state they even go as low as 0.05mg/ml of BSA for standard curve. Is it ok to go that low and will this kit have a problem detecting it?
Edited by azrael201, 01 February 2011 - 08:25 AM.