LAB #5: Bradford Assay Worksheet 1. Complete the tables below: BSA STANDARD CURVES You can calculate how much BSA is actually being read in the
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The black cells are then data we collected from the experiment. Not sure if all I need to do is transfer that data to the first question or if they are asking for a different calculation

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LAB #5: Bradford Assay Worksheet 1. Complete the tables below: BSA STANDARD CURVES
You can calculate how much BSA is actually being read in the spectrophotometer and write it down in the worksheet below. In other words, you prepared a 1000 p.L solution of the standard protein, but you
only pipetted 5 pL into each well to read. How much protein is actually in the well? {2 pts} [BSA] Volume lgG
{mg/ m L) stock (ml) Well if A595 Well # A595 Average A595 0-0 A1 31 A1, 31
0-1 A2 32 A2, 32
0-2 A3 33 A3, 33
0-3 A4 34 A4, 34
0-4 A5 35 A5, 35
0-5 A6 33 A6, 36
0-6 A7 37 A7, 37
0-7 A3 33 A8, 33
0-8 A9 39 A9, 39
0-9 A10 310 A10, 310 1.0 All 311 A11, 311

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Materials
NOTE: Make sure you have all the materials ready before you start pipetting-
Each group of 4 will need:
1 clear 96-well plate
1 vortex mixer
22 16 x 100 mm glass test tubes
Test tube racks to hold the glass test tubes
15 mL of "Bradford Reagent" (obtained from Pierce Coomassie Protein Assay Kit, Cat#23200
from Fisher Scientific)
One P20, one P200, and one P1000 micropipettor
50 ml Sodium Phosphate Buffer, pH7.4 (same buffer used to homogenize your algae)
Provided by TA: One tube with stock BSA at 20mg/ml (in SPB, pH 7.4)
Provided by TA: One tube with stock IgG at 20mg/mL (in SPB, pH 7.4)
Procedure
1. Pick up an algal extract tube and thaw your frozen protein samples from the last exercise.
Preparing the Standard Curves: BSA and IgG
2. While your protein samples are thawing, pick up one tube of each standard protein (BSA at
20 mg/ml and IgG at 20 mg/ml) and the Bradford assay reagent. Using 22 glass test tubes,
prepare 11 dilutions of these protein standards. The information below will aid you in the
preparation of your standards.
NOTE: Dilution calculations (tables below) should be done prior to lab and shown to instructor
prior to start of experiments.
Use the sodium phosphate buffer (SPB, pH 7.4) to prepare your dilutions. In a 1.0 mL (1000 ul)
total volume, you will prepare:
i. BSA, 0.0 to 1.0 mg/ml dilutions (0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg/ml]
ii. IgG, 0.0 to 1.5mg/ml dilutions (0.0, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, and 1.5 mg/ml)
* Note that the 0 Tube (0 me/ml of protein) should only include SPB.

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BSA Dilutions (fill in with the correct volumes to generate the required concentrations)
Tube I
1
2
3
4
5
6
7
8
9
10
11
OSA (mg/ml]' 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
SPO (AL)
USA stock (uL)
Total Vol. (PL)
1000 1000 1000 1000 1000
1000 1000 1000 1000
1000
1000
"Final BSA concentration in ma/mL Note that the stock concentration is 20 me/ml
IgG Dilutions (fill in with the correct volumes to generate the required concentrations)
Tube il
1
2
3
4
5
6
7
8
9
10
11
IgG (mg/ml)*
0.0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.5
SPO (AL)
gG stock (AL)
Total Vol. (AL) 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
"Final IgG concentration In me/mL. Note that the stock concentration is 20 mg/ml
3. Once you have prepared each of the above dilutions in a small glass test tube, thoroughly
vortex each tube at a low level (e-g. 4) to avoid spills.
4. Pipet 5 UL of the dilution in tube #1 (0.0 mg/ml) into well A1 of 96 well plate. Pipette 5 ul of
the dilution in tube #12 (0.1 mg/mL) into well A2 of the 96 well plate. Note that you do not
need to change your pipette tip if you are going from the lowest BSA concentration to the
highest concentration. Change your tip when you switch between standards or samples.
Continue transferring S ul of each of the dilutions into the appropriate wells based on the
diagram below.
[ Low] -
[High]
1
2
10
11
Samples (in duplicates]
$1 - 5 ul of tube "E" extract
OOOOOOOOO
52 - 5 ul of your unbound
"purified" protein
53 = 5 ul of your bound "purified"
protein
Samples
GS ) S

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5. Once all your standards and samples have been added to the wells, add 195 pL of the
Bradford Reagent (Coomassie Brilliant Blue dye solution) to each well, starting from the
lowest concentration of the protein and moving to the highest. Ideally, you want to
dispense the Bradford reagent as quickly as you can so that each sample will be exposed to
the dye for approximately the same amount of time as the absorbance of the protein-dye
complex will continue to increase over time.
NOTE: To minimize plastic waste here, you can, use the some pipette tip for all wells of BSA,
a second tip for all wells of IgG, and a third tip for your samples. Move from low to high
concentration of the protein avoiding touching the protein solution already in the well, If you
do touch the protein in the well, you should change your tip.
6. Incubate the plate at room temperature for 15 minutes.
Data Collection
7. After incubation, notify your instructor so they can show you how to collect the data using a
plate reader.
8. Save your results in a CSV file format. Use a USB from the instructor to transfer the file to
your computer and upload the file to a spreadsheet (e.g. Excel or Numbers). Alternatively,
your instructor will load the class files onto Canvas.
9. Record the absorbance values and additional information for your samples and standards
below or in an excel file.

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