Although the specifics for the assays will vary by protein, it is recommended to start with the most concentrated wild-type protein solution and crudely measured out reagents. Making stock solutions, and the order in which components are mixed together, is of utmost importance. It is prudent to keep separate stock solutions of every component until the behavior of every component is known (Is it temperature sensitive? Precipitate over time? Does it kill protein? Or, only last one class period?). Mixing reagents together in one big stock solution at the beginning of running assays sacrifices much of the control in the experiment.
Here are some tips for finding a reproducible assay method:
- First, how is the reproducibility? Can the assay be repeated three times in a row and yield the same initial reaction velocity? Test this with the crude assay mixture.
- Adding too much enzyme can finish the reaction before mixing is complete (i.e., the zero baseline should change).
- Putting the cuvette in the wrong way or using the wrong type of cuvette can frustrate even the smartest of groups.
- Make sure the pH is correct and that it does not change substantially during the reaction.
- When mixing components together for assays, try not to use volumes less than 10 µl. If need be, make a more dilute stock solution of enzymes and substrates to accomplish this goal.
- Some reagents decompose (over the course of a lab period) if placed in the assay buffer. Instead, make a 100x stock solution of that reagent and keep it in water or storage solvent on ice.
- The enzyme may need to warm up to room temperature before it works well.
- Improper mixing makes the data look groovy and not linear.
- Determine the longevity of the enzyme—does the enzyme die quickly? Will it have the same activity next week?
Note: the following example is not an assay that is appropriate for your enzymes-rather it is just an example of an assay that can be monitored spectrophotometrically!
Here is an example for a 1 mL enzyme assay reaction that needs Zn+2 ion. 10x stock solutions of all the components (dissolved in assay buffer) can be made.
Place 780 ml of assay buffer into a cuvette (stored at room temperature)
Add 100 ml of ZnCl2 solution (10x, in assay buffer) (stored at necessary temperature)
Add 100 ml of substrate solution (10x, in assay buffer) (stored at necessary temperature)
Place the cuvette into the instrument and zero it. It may be useful to run the instrument through a full data collection set to see what happens. Nothing should happen, since the enzyme has not been added yet. If something is changing, this is the background reaction and it is important to monitor.
Add 20 ml of the most concentrated wild-type enzyme solution (stored on ice)
Quickly mix by pipetting with a P1000 pipette.
Is there activity? Yeah! Keep in mind you will want to run a control reaction without enzyme to make sure that the data looks different.
Now modify the assay so that all the needed kinetic constants for publication can be gathered and the ncAA-mutant enzymes can be tested. Streamlining the assay before taking serious measurements can be useful—aspects such as temperature control, taking data in triplicate for error bars, and other aspects are good to outline before taking a series of measurements for all of the enzymes.
Deep Thoughts on “How do I write a good paper? What do I need for publication?”
By the end of this term, you will hopefully have been exposed to a significant body of literature surrounding your protein of interest. Now, you have to prepare your research in a manuscript. Think back to some of the papers you read. What did you like about these papers? What did you dislike about these papers? Perhaps you liked when the authors told a story that drew you in or when the data was presented in a clear and concise manner. You can use your own experience as a reader to guide your writing.
Aside from creating a manuscript that people will actually enjoy reading, it’s also important to consider the utility and reliability of your work. Much like you did at the beginning of this term, when embarking on a new project, pursuing a new area of research, or investigating a new technique, scientists tend to scour the existing literature to find out what has been done and what is already known. As such, it’s important that these papers include the information required to repeat the experiments and replicate the results.
Any reader will also want to be able to assess the quality and meaning of your work. For something like a kinetics assay, it’s important to carry out multiple trials (ideally, triplicate) and to include error bars. This allows the reader to decide if your results were just a fluke or if they were consistent and meaningful.