Lab 2: Protein Extraction and Quantification

RIPA Buffer

• chemical method by which protein may be extracted from a cell

  • disrupts the phospholipid membrane and extracts the protein

• RIPA contains the detergent Triton X-100

  • also includes protease and phosphatase inhibitors to ensure that the extracted proteins do not denature

Bradford Reagent

• when the Bradford reagent dye binds to the proteins within the sample, there is a colour change from brown to blue.

  • the colour change is measured via spectrophotometer

• the bluest samples are indicative of the largest reaction, and therefore the greatest amount of protein (and a high absorbance rate)

BSA

• bovine serum albumin

• our assay used BSA as a generic protein standard to which the unknown samples can be compared.

  • We typically make a dilution series with the BSA (ranging from 0.0 – 1.0 μg/μl).

• +, linear relationship between [BSA] and absorbance

Standard Curve

• +, linear relationship between [BSA] and absorbance

• the line of the “standard curve” or a “calibration curve” can be used to determine the protein concentration in an unknown sample

Ways to Lyse Cells/Extract Cellular Components

• physical methods: uses external forces to break down the membrane to release the cellular components

  • liquid homogenization: shears cells by forcing them through a narrow space

    • can be done using a plunger and vessel, or an automated fashion

  • sonication: machine which uses pusled, high frequency sound waves to agitate and lyse cells

    • soundwaves are delivered using an apparatus with a vibrating probe that is immersed into the cell suspension

  • manual grinding: works well to isolate proteins from plant cells

    • tissue is frozen in liquid nitrogen, then smashed and releases proteins

• chemical methods: use of detergents/hypotonic solutions to disrupt the phospholipid membrane and extract the protein

  • RIPA buffer

PLS MR

• physical: liquid homogenization, sonication, manual grinding

• chemical: RIPA

Bradford Assay

• quick/easy way to find the total concentration of protein in a solution

• simple test; when the Bradford reagent dye binds to protein, there is a colour change from brown to blue. The intensity of the blue colour is measured using a spectrophotometer

• set up:

  • preparation of 5 cuvettes:

    • 0 µl cell lysate, 100 µl PIPES buffer

    • 5 µl cell lysate, 95 µl PIPES buffer

    • 5 µl cell lysate, 95 µl PIPES buffer

    • 10 µl cell lysate, 90 µl PIPES buffer

    • 10 µl cell lysate, 90 µl PIPES buffer

  • to each cuvette, 5ml of Bradford Reagent is added

  • wait 10 minutes

• spectrophotometer

  • blank the machine

  • take absorbance readings

Making Dilutions (given a stock solution)

Step 1

• 1 hour prior to lab, flaks containing CHO cells at various temperatures were placed either in the fridge 4o C (cold shock), 37o C (negative control) or a 44o C (heat shock) incubator

Step 2

• Pipette the old media with a PipetBoy and transfer to a Falcon tube

Step 3

• Use a P1000 micropipette to add PBS to the flask, and swirl over the cells. Wait 5 seconds, then dispose of the PBS into the Falcon tube

Step 4

• Add trypsin to the flask of cells and incubate for 3 minutes. Tap the side of the flask to help dislodge the cells

Step 5

• Add fresh media in with the cells and aggressively mix by pipetting up and down

Step 6

• Transfer everything from the flask into the Falcon tube

Step 7

• Centrifuge the Falcon tube to pellet the cells, then dispose of the supernatant

Step 8

• Add RIPA buffer, and sit tube on ice for 12 minutes

Step 9

• Microcentrifuge, and divide…

  • supernatant tube A, set on ice

  • supernatant and Sample Buffer tube B, place in freezer

Step 10

• Divide the tube set on ice into 4 cuvettes, and leave 1 empty for the control. To each cuvette, add PIPES buffer, and Bradford reagent

Step 11

• Stretch parafilm over the top of each cuvette, invert several times to mix, and wait 10 minutes

Step 12

• Blank the spectrophotometer, and record absorbance values for the 4 treated cuvettes

Step 13

• In excel, plot the BSA concentrations to the absorbance readings, and calculate the equation of the line