[Arkcon]

I'm just completely confused at what could cause this

Really?  Your "10 ml" samples look give essentially the same number in each case.  Those look to me like you are below the ability of the system to detect an absorbance.  How do the "10 mL" samples compare to a blank, just coomassie blue and water?  If there's no difference, then you can't use your procedure at that level.  Your "25 mL" seem to take a sudden drop as you go to lower levels, even those samples may not be concentrated enough for your system you quantitate them.

Any of a number of sample handling problems could also have affected that sudden drop.  Are these serial dilutions?  Or did you prepare each one individually?

[orgo814]

These are serial dilutions. I picked the wrong max absorbance wavelength to blank and work at. Could this have caused this?

[Corribus]

I may have solved my problem. Did research and the coumassie I had was the reddish kind with max absorbance at 470 and I was doing mine at 595.

This would certainly be a problem, which is why I suggested taking an entire spectrum.

[orgo814]

Well I will test it out when back in lab but I have to think this is it. Thanks for your help I appreciate it.

[orgo814]

I diluted the coumassie before using it but I still think the max wavelength is still lower than what I set it as. There are multiple coumassies.

[Irlanur]

Some comments

-please dont only use r^2 as the only factor to judge your curve. there are bad curves with good r^2
-ALWAYS look at the absorption spectrum of your compound if you do a new experiment. it takes no time.
-look at your blanks

[orgo814]

I keep repeating it and getting same r^2 (around 0.85). Question: Are some standard curves going to have a bad r^2 and still be what would be acceptable for them? I was looking at some standard curves for BSA and not all of them are linear. I guess it depends on my concentrations?

[Arkcon]

I keep repeating it and getting same r^2 (around 0.85). Question: Are some standard curves going to have a bad r^2 and still be what would be acceptable for them? I was looking at some standard curves for BSA and not all of them are linear. I guess it depends on my concentrations?

You haven't answered some of the questions I asked, and my concerns were repeated by Irlanur:

Are your results significantly different from a blank?  If not, you don't have enough sample to run your assay.  The "10 ml" samples look all the same, they have a quite good r2 value, for all the wrong reasons.  unless your sample isn't even reaching the beam of the instrument -- unless this "10 ml" sample isn't "10 mls of sample diluted to the same volume as the 25 mL sample"  We're not there, so its up to you to give us all the information on your assay:  what is the sample, what is its initial concentration, how are you diluting it, and with what.  What is your coomassie blue solution, how much of it is in each sample, what are you diluting your final samples with, and what wavelengths are you using.

[orgo814]

The sample is BSA. I make up solutions with the concentrations I listed earlier (through serial dilutions). I took 15uL of each solution made up and added it to 1 mL of my coumassie reagent. My coumassie reagent was made from 1 mL of the coumassie and 4 mL of nanopure water. The coumassie was actually red but when diluted had a greenish tint. My blank was greenish while the others were a brilliant blue (after addition of the coumassie reagent to 15uL... I think it turns that blue color when it binds to the protein).

[orgo814]

Also, I worked at 595 nm.

[Arkcon]

So, given the shift in wavelength given below, blanking isn't as useful with coomassie blue as in other situations.  This is problematical, but not a disaster.  You mention working with a 1 ml sample size, that should put the fluid level in the beam path, but do be sure.  For example, a serial dilution of the Coomassie Blue, without any sample, should show a linear absorbance, even if the wavelength with maximum absorbance isn't the same with protein bound.  You could try evaluating that, to conform the instrument is working correctly and you're preparing things correctly.

[orgo814]

I mean, I did it a second time and had my absorbances 0.778, 0.698, 0.533, and 0.328, respectively. Which is improved. There's a downward trend which is what I am looking for but my linearity is still not great.