Bradford Protein Quantitation Assay (Time: 20 minutes)

1

Entered testing area wearing closed-toed shoes, then donned proper PPE: glasses/safety glasses/goggles and gloves (lab coat is optional).

2

Labeled two empty microcentrifuge tubes for the sample dilutions (for example, 1/50 A and 1/50 B, or A and B)

3

Pipetted 2 µl of sample A into the microcentrifuge tube labeled 1/50 A. *Judge verified competitor (i) selected 20 µl micropipet, (ii) set micropipet to correct volume, (iii) used a clean pipet tip, (iv) used a proper pipet tip, and (v) transferred liquid without air bubbles or losing sample in the transfer process

4

Pipetted 98 µl of 1x PBS into the microcentrifuge tube labeled 1/50 sample A. Judge verified competitor (i) selected (20-200 µl micropipet, (ii) set micropipet to correct volume, (iii) used a clean pipet tip, (iv) used a proper pipet tip, and (v) transferred liquid without air bubbles or losing sample in the transfer process

5

Mixed well by pipetting, flicking, or vortexing.

6

Pipetted 2 µl of sample B into the microcentrifuge tube labeled 1/50 B. *Judge verified competitor (i) selected 20 µl micropipet, (ii) set micropipet to correct volume, (iii) used a clean pipet tip, (iv) used a proper pipet tip, and (v) transferred liquid without air bubbles or losing sample in the transfer process

7

Pipetted 98 µl of 1x PBS into the microcentrifuge tube labeled 1/50 sample B. Judge verified competitor (i) selected 20-200 µl micropipet, (ii) set micropipet to correct volume, (iii) used a clean pipet tip, (iv) used a proper pipet tip, and (v) transferred liquid without air bubbles or losing sample in the transfer process.

8

Mixed well by pipetting, flicking, or vortexing

9

Labeled two cuvettes: Sample A and Sample B (or just A and B)

10

Pipetted 20 µl of the 1/50 diluted samples into the corresponding cuvettes. *Judge verified competitor (i) selected 20 µl micropipet, (ii) set micropipet to correct volume, (iii) used a clean pipet tip for each liquid, (iv) used a proper pipet tip, and (v) transferred liquid without air bubbles or losing sample in the transfer process.

11

Labeled eight cuvettes for the protein standards: Blank, 0.125, 0.250, 0.500, 0.750, 1.000, 1.500, 2.000

12

Pipetted 20 µl of 1x PBS into the cuvette labeled Blank. *Judge verified competitor (i) selected correct micropipet (20 µl), (ii) set micropipet to correct volume, (iii) used a clean pipet tip, (iv) used a proper pipet tip, and (v) transferred liquid without air bubbles or losing sample in the transfer process.

13

Pipetted 20 µl of each protein standard into a corresponding cuvette. *Judge verified competitor (i) selected correct micropipet (20 µl), (ii) set micropipet to correct volume, (iii) used a clean pipet tip for each standard, (iv) used a proper pipet tip, and (v) transferred liquid without air bubbles or losing sample in the transfer process.

14

Added 1 ml of the 1x Bradford reagent to all ten cuvettes. Mixed well by pipetting up and down. *Judge verified competitor (i) used the correct micropipet (100-1,000 µl), (ii) set micropipet to correct volume, (iii) used a fresh pipet tip for each sample, (iv) used a proper pipet tip, (v) transferred liquid without air bubbles or losing sample in the transfer process, and (vi) pipetted up and down gently to mix (no liquid sucked into the barrel of the micropipet, for example).

15

Verbalized “cuvettes incubate at room temperature for 5 minutes”.

16

After visually comparing the cuvettes containing samples to the cuvettes containing the protein standard, verbalized an estimated protein concentration of the sample

17

17. Cleaned work area:

a. Disposed of used pipet tips, microcentrifuge tubes containing the 1/50 dilutions, and cuvettes in waste receptacle.

b. Cleaned work area with surface disinfectant.

c. Removed PPE. d. Washed hands or used alcohol-based hand-rub for hand hygiene.