Protein quantification using Bradford Assay with Coomassie

Overview

Protein binds to the Coomassie Plus reagent and causes a shift in signal from 465 to 595 nm with a simultaneous color change of the reagent from green/brown (or red/brown) to blue. Pierce Coomassie Plus Protein Assay (Cat # 1856210) has a detection range of 1µg/ml to 1µg/ml of protein. The protein of interest must have a molecular weight greater than 3,000 daltons to be detected by the assay. For peptides smaller than 3,000 daltons, try using Fluoraldehyde Reagent Solution (Product # 26025), which will fluorescently detect amino groups on small peptide. The time the Coomassie reagent sits with the sample will affect the reading. Try to keep all samples on ice until shortly before measuring the samples. The 96 well plate should be at room temperature when taking measurements. Coomassie reagent should be mixed before every step. Coomassie + Protein solutions should sit for 10 minutes RT before taking measurements. If they sit for longer than 30 minutes they start to form a precipitate, so they need to be mixed repeatedly. The standard Bradford assay is accurate fµcro; µg/ml to 1,µg/ml when using BSA as a standard, although it is possible to measure up tµcro; µg/ml. Alternatively you can use the "micro" Bradford assay for samples that are from 15 to 125 µg/ml (see below). To get an idea of how concentrated your protein sample is, make a quick sample and compare to highest and lowest standards, to make sure your sample is in the range of the standards.

Protocol

• Prepare the computer program
• Open Gen5 software
• In the Task Manager window select "Experiments" then select "Create using an existing protocol"
• In the window that opens up, select "My Documents" then "Starr lab" then "Epoch protocols".
• Open the "Coomassie Plus Bradford Protein Assay" protocol. (Notes on how I set up this protocol are at the end of this document)
• Open the Protocol options by clicking on "protocol" in the left window.
• Open "Plate Layout". The Plate layout has A1 B1 with blanks, A2—A9 and B2—B9 with the 8 standards, and the rest of the wells with samples. Note that the plate is set up to have two replicates of every blank, standard, and sample.
  • To delete samples, click on the sample in the left window (eg. SPL11 (x2)) and press the delete button.
  • To add samples, First change the Replicates to 2, then click in the first open well (note the pointer changes to a pipette) and both the well and the one below will be added as the next sample. This assumes you have the Auto Select Next ID box checked.
• When you have the plate layout finished click the OK button. The window should close
• Click on "Plate 1" in the left window. If you want to label the plate, click on the "Information" icon and enter a Plate ID, then click OK. If you want to label the samples, click on the Sample IDs and fill in the Sample IDs in the list, then click OK.
• The program is now set up and ready to measure the absorbance at 595. Proceed to preparing the standards and samples.

• Prepare the 8 protein standards
• Prepare standards in 1.5 ml microfuge tubes using BSA from NEB at 10 mg/ml.
• Place 25 µl of H2O in tubes 1 through 8.
• Add 25 µl of BSA (10 mg/ml) to well A8.
• Serially transfer 25 µl into tubes 7, 6, 5, 4, 3, and 2.
• Leave tube 1 with just water, no protein.
• Mix well in between each serial transfer.

Standards set up

• In a second set of 8 tubes place 490 µl of Coomassie reagent.
• Transfer 10 µl of standard 1 from tube 1 to the corresponding tube 1 with Coomassie reagent (1:50 dilution).
• Repeat for all 8 tubes.
• Each tube will have enough standard to place two replicates of 200 µl each in a 96 well plate.

• Prepare unknown samples.
• In 1.5 ml microfuge tube mix 10 µl of protein sample with 490 µl of Coomassie reagent and mix well.
• Load blanks, standards, and samples into 96 well plate (make a map if you need one). There will be two replicates for all samples. Each replicate will be in the matching row below. So A1 and B1 will contain water only (Blank) and A2—A9 and B2—B9 will contain standards 1—8. A10 and B10 will contain sample 1, A11 and B11 will contain sample 2, etc. From the standard tubes and sample tubes above transfer 200 µl into the two replicate wells in the 96 well plate.

• Read the plate
• Select the Green arrow button to read the plate.
• Fill in any prompts if you want, or just leave blank and click the OK button.
• The Plate 1 window will open and the Load Plate window will open instructing you to place the plate on the carrier. Place the plate on the carrier and select OK.
• The Epoch machine will read the absorbance at 595 in the wells indicated in the protocol.
• When finished the program will eject the plate.
• If the standard curve is not logical a Calculation Warnings window will appear telling you that the curve is messed up. If this occurs, your standards are not good and you need to prepare them again.
• If you have multiple plates, repeat the process starting by selecting the green arrow. A new plate will be added and read.
• Save the experiment by clicking on file → save, then navigating to your folder under My Documents → Starr Lab → your folder → your subfolder.
• Name the file using the date "yrmoda" e.g. 111202 and whatever descriptive words you want.
• Transfer the data to an excel spreadsheet by selecting Plate → Export (or use the button with the blue arrow on the white sheet).
• Excel will open and the data will be copied to a worksheet called Book1.
• Save the worksheet with the same name as your experiment in the same folder.
• The "Mean" column will give you the concentration of each sample.
• Print out the table for your lab book.
• Quit the Gen 5 program. If you changed the plate layout or anything else in the program a window will pop-up asking if you want to Update, Save As, or Ignore the changes. Generally you should select "Ignore" so the protocol will be the same for everyone.

• Measuring protein concentrations in the 15 – 125 µg/ml range.
• Prepare standards in 1.5 ml microfuge tubes using BSA from NEB at 250 µg/ml
• Dilute the 10 mg/ml BSA stock by adding 25 µl of 10 mg/ml BSA to 975 µl H2O
• Place 250 µl of H2O in tubes 1—8
• Place 500 µl of 250 µg/ml BSA in well A8, then serially transfer 250 µl into wells A7—A1.

Micro Standards set up

• In a second set of 8 tubes place 250 µl of Coomassie reagent,
• Transfer 250 µl of standard 1 from tube 1 to the corresponding tube 1 with Coomassie reagent (1:2 dilution).
• Repeat for all 8 tubes.
• Each tube will have enough standard to place two replicates of 200 µl each in a 96 well plate. Proceed as above.

Notes on Coomassie protocol in Gen5:

• The Coomassie protocol is as follows: Procedure: Read (A) 595. This is the correct absorbance for the Coomassie reagent
• Plate Layout: Replicates = 2, the replicates are in vertically adjacent wells
• Auto Select Next ID is checked
• Data Reduction: Transformation subtracts the average of the two blank wells from the average of all the other replicates.
• Standard Curve uses a Nonlinear 4 parameter fit model Y=(A-D)/(1+(X/C)^B)+D equation with an Extrapolation Factor of 1.1 (this just allows to assign concentrations to unknowns that are a little higher or a little lower than the largest and smallest standards.) This number can be between 1 and 3. 1 means there will be no extrapolation.
• Data Out is Calculate Concentrations Report/Export Builders: The only changes to the default output file is that the General format is a Row-wise table, not a Matrix and the Include curve/scan pictures box is checked.