Lab 10: PROCEDURE

Assay overview and technical considerations

Here is a link to two videos created by one TAs that provide an overview of the HCAII kinetics assay:

Lab 10 assay overview video

Lab 10 assay set up details video

Assembling and running a kinetics assay

This video demonstrates how to:

• set up a kinetics assay in a cuvette
• run the spectrophotometer
• find your data when the read is finished

Setting up the spectrophotometer

• Turn on the Shimazdu Spectrophotometers and allow it to warm up (this takes about 15 min.)
• This is a dual-beam spectrophotometer. It has two slots for cuvettes so that it can measure sample and blank at the same time.
• Set the following parameters on the Shimazdu Spectrophotometers:
  • From the main menu, select Kinetics (# 4)
  • From the next menu, select 1 cell kinetics/λ (#1)
  • Measure time = 40 seconds
  • Next hit the “Goto WL” button. Then type in 405. The spectrophotometer should change the wavelength to 405 nm.
• Note there are also spectrophotometer instructions in the Appendix (link opens in a new tab): Appendix 5: Operation of the Shimadzu spectrophotometers

Pilot reactions

The purpose of a pilot reaction to is be sure that your experiment is working (i.e. you are able to measure something), and that your experimental conditions result in a signal within the range that the spectrophotometer can reliably read. This has been somewhat optimized over several years of 551 labs, but the exact optimal conditions may vary a bit depending on your protein sample and your mutation. You will test one condition to start, and if the resulting signal falls within a good detectable starting range, you can proceed with the experiment using those conditions. If, however, the signal is too high, you will need to decrease the protein concentration used. Conversely, if the signal is too low, you will need to increase the protein concentration used.

• The pilot reaction should contain:
  • A final protein concentration of 0.1 µM
  • 20 µL of 50 mM PNPA (for a final concentration of 1 mM)
  • Buffer to a total of 1 mL
• We recommend assembling the reaction directly in the cuvette (this also saves plastic). Add buffer first, then protein. Always add PNPA last, when you are ready to measure.
• Once the PNPA has been added to the reaction you will need to act quickly. Make sure your reaction is well mixed before measuring  – mix by gently pipetting up and down with a p1000.
• Your reaction should be in the front slot of the spec and your blank should be in the back.
  • The blank should contain protein buffer and the [PNPA] you are testing, but no protein.
• Once both samples are in the spectrophotometer hit “START” immediately.
• Once the reaction has finished, look at the graph. If the line runs off the top of the graph (i.e. the absorbance went above 2) within the first 10 seconds, the reaction rate is too fast and you need to reduce the amount of enzyme you are using. Next, press the “Datalist” button to see the slope of your reaction. Your slope should be around 0.1. If it is not, increase or decrease the amount of enzyme you are using. (If you are in the correct range, you can repeat the assay twice more and use this as one of the substrate concentrations in your data set.)
• Important notes:
  • If you do not have enough enzyme to do the remaining assays, reduce the amount of enzyme you are using as much as possible. Alternatively, you may have to collect fewer data points. Discuss with a TA if you are unsure what to do.
  • Be sure to write down the slope of the line (i.e. ΔA/min) – you will use this data to calculate the reaction rate. The spectrophotometer will not save your data.
  • It is possible that you will need to use different protein concentrations for wt vs. mutant in this assay. Pilot experiments should be conducted separately for wt and mutant.

• Once you have determined the amount of enzyme you will use for each reaction, plan the rest of the assays.
• Since you are going to be calculating the K_M and V_max for your mutant and wild-type enzymes, you will want to test a range of PNPA concentrations above and below the K_M. In lab you will work with your partner to find the published K_M. You can then select 8 substrate concentrations to test.
  • Note that for technical reasons you cannot test [PNPA] >5 mM.
  • You will need a separate blank for each [PNPA].
  • You will run each [PNPA] in triplicate. It is possible to use the same blank for all three replicates as long as the PNPA in the blank has not hydrolyzed too much (i.e. turned yellow); however, it is challenging to move quickly enough (especially at high [PNPA]), and you may get cleaner data by making a fresh blank for each measurement.

• At the end of lab, SAVE YOUR PROTEIN SAMPLES AT 4 ºC.