4 replies to this topic

[azrael201]

Hi guys,

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?



Muchos gracias!

Edited by azrael201, 01 February 2011 - 08:25 AM.

[mdfenko]

azrael201, on 01 February 2011 - 08:22 AM, said:

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?

the volume in the cuvette should make the height of the solution above the light beam from the spectrophotometer. dilution is not always a good answer, it will reduce the sensitivity of the assay. either find cuvettes that match the genova volume/height or scale up the assay.

Quote

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?

the microplate assay is a scaled down version due to the lower volume of the plate wells. protein concentrations of the standards will be the same but the amount of protein will be different. as long as you maintain the proper ratios then scaling will be valid.

Quote

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.

quartz cuvettes are good for the entire range of the spectrophotometer, including uv, they are not uv only. they are not disposable and require proper cleaning after use. glass cuvettes are generally good for the visible range. glass is also not disposable and require proper cleaning after use. polystyrene is good only for the visible range and is disposable (some acrylic disposable cuvettes are okay for uv).

Quote

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?

do what the manual says. you can not properly evaluate samples outside the linear range (at least, not without some fancy algorithm) even if they are within the range of your standards. some proteins give different response than others. this is more pronounced with the bradford protein assay where bsa gives ~2x the response of igg. the dc assay (based on the lowry assay) is less variable with different proteins so the linear range for bsa and igg should be about the same. you should, however, determine which standard most closely resembles the response with your protein (i used the fluorescamine assay to make the comparison, the kjeldahl, or micro kjeldahl, is also good for confirming the result) and continue with that standard.

Edited by mdfenko, 02 February 2011 - 11:21 AM.

[azrael201]

wow thanks mdfenko. great read.

now that you mention it i don't know if it is quartz or glass but if the postdoc thinks it is only for UV i'm guess it's safe to say it's probably quartz. how do i properly clean them? I've read conflicting things but I would assume ethanol is sufficient; however, i think i did read how ethanol could destroy the special coating and to use acid instead?

in between readings i have been just aspirating with a glass pipette is that bad or should i be rinsing with something like distilled water?

also regarding your last statement does that mean i should replicate the linear curve with my sample in order to determine how comparable they are?

Edited by azrael201, 02 February 2011 - 12:26 PM.

[mdfenko]

quartz and glass cuvettes can be routinely cleaned by flushing with diwater and then a drying agent (ethanol or acetone). we use a cuvette washer.

plastic cuvettes can be cleaned with water and alcohol.

when washing after a dye binding assay (eg-bradford) wash with alcohol, water, alcohol (disposables are better for these, if you use quartz or glass then you have to ensure that cleaning is thorough because the dye tends to bind to the cuvette).

we used to soak the quartz and glass cuvettes in nitric or chromic (later, nochromix) acid to remove any residues that may be left behind, then washed. you can also soak in a solution of terg-a-zyme.

i am unaware of any special coatings on cuvettes which can be washed off with ethanol.

if we use a glass pasteur pipet with a plastic cuvette then we would add a soft plastic tip (made from microline tubing so that the tip can be pulled into a taper) so that it won't scratch the walls (especially the windows).

as for the standard curve, it is optimal if you can use your purified and quantified protein as a standard to generate it. under less ideal circumstances you can determine which routine standard (bsa, igg,...) most closely resembles your protein in its response to the assay you use by using a different assay (this can be tedious and should only be required once for each protein that you prepare).

Edited by mdfenko, 04 February 2011 - 11:29 AM.

[azrael201]

where can i read more about protein assays and designing these curves?

i'm using the Biorad DC kit and although it says to use either BSA or IgG i thought i read that it can make a difference. I have been using BSA and i can't seem to get reliable readings any log lower than 0.1

also interpreting the curve am i suppose to find the linear portion so that means if i have multiple points on the standard i should pick the points that form the linear portion and fit the line?