Skip to content

Latest commit

 

History

History
78 lines (54 loc) · 4.24 KB

kinetic_assay.md

File metadata and controls

78 lines (54 loc) · 4.24 KB
Raw

Assay to determine kinetic parameters of β-glucosidase B

This assay assumes that you have followed the protein production and purification steps in production.md in this repository.

Mise en place

  • waste bucket
  • pipet reservoir basin
  • P200 and tips
  • Multi-channel P50 and tips
  • Multi-channel P300 and tips

Software

  • purified protein from day 4
  • substrate stock (you will need 2 mL)
  • protein buffer
  • 2 mL tubes
  • 1 strip of PCR tubes

Instructions

Before you begin

  1. Prepare your mise-en-place (put everything in place)
  2. Thaw your substrate stock and vortex if necessary until completely clear (no precipitate), it may look a little bit yellow due to background pNPG hydrolysis, this is normal

Perform a qualitative test of enzyme activity for each sample

  1. In a PCR strip, aliquot 9 µL of substrate stock, 1 well per mutant
  2. Aliquot 1 µL of each mutant, 1 well per mutant
  3. Incubate at room temperature for 5 minutes
  4. If the sample turns yellow:
    • make a 1/100 dilution in a fresh tube: mix 10 μL purified protein and 990 μL wash buffer
  5. Else:
    • make a 1/10 dilution in the fresh tube: mix 100 μL purified protein and 900 μL wash buffer

Use the diluted purified protein for the remainder of the procedure. The undiluted stock can be used to run a gel and quantitate protein concentration by A280.

Prepare a substrate plate

First, make the substrate plate. You can use any convenient plate for this, the $1 Costar full-area plates are best.

  1. Using a single-channel pipet, dispense 150 μL of substrate stock into row A (12 wells)
  2. Using a multi-channel pipet, dispense 112.5 μL of protein buffer into rows B, C, D, E, F, G, H (the rest of the rows)
  3. Using the multi-channel pipet, remove 37.5 μL from the first row and add to second row, and mix three times
  4. Repeat this procedure for the second, third, forth, fifth, and sixth rows
  5. Remove 37.5 uL from the seventh row, and throw away the liquid with the tips. Do not touch the last row in this step
  6. Verify that every well in the plate is the same volume (112.5 µL)

Prepare a protein plate

Now that you have created the gradient of substrate concentrations in the substrate plate, aliquot your proteins into the protein plate

  1. For each mutant, aliquot 25 µL of diluted purified protein into three columns of the plate (24 wells)

Initiate assay and collect data

To initate the assay, transfer 75 µL from each well in the substrate plate into the assay plate containing your aliquotted protein. Before you begin the instructions below, set the plate reader to monitor absorbance at 420 nm every minute for 60 minutes.

  1. Using the multichannel pipet, transfer 75 µL from the bottom row of the substrate plate into the bottom row of the assay plate
  2. Repeat for the rest of the rows (bottom up)
  3. Place the plate on the prepared plate reader and press Enter (run time: 1 hour)
  4. Important: fill out the "Information" panel (on the Gen5) with your name, the mutants you are testing, and the dilutions that you used. For example:

Alex Carlin. Cols 1-3: WT @ 100X, cols 4-6: E164A @ 10X, 7-9: I300L @ 100X, cols 10-12: E353A @ 10X

Advice: Now is a good time to check to make sure you have entered your finished production data into the report sheet.

Data analysis

  1. Once the assay has finished, remove your plate and examine your data. Note: make sure that the Gen5 is set to calculate rates ("MaxV [420]") based on at least 10 points, and that the rates are reported in units of OD/min. These settings can be found under "Data Reduction > Calculation Options"
  2. Visually check and assess R-squared values for the 96 rates on your plate. R-squared values closer to 1 are better. Mask points as necessary to compensate for detector limit (it can't read above OD 3.75)
  3. Use the Bagel project web app to fit your observed rates to the Michaelis-Menten equation
  4. The web app will return functional parameters for your mutants
  5. Visually check the fits and reported errors (should be less than 50% error, but this is not the only criteron you can use to determine if a fit is good)
  6. Enter your production data on the production sheet of the report
  7. Enter your assay data on the assay sheet of the report