Purchasing of reagents:

Our laboratory class typically consists of 25-30 students, or 13-15 student pairs. Most of the experiments are best performed by student pairs. 

We typically order reagents that are labile or expensive on an as needed basis, and the quantities of these reagents listed at the back of the Chapters reflects our typical class size. Reagents that are inexpensive and stable are usually ordered in excess, and thus instructors should not blindly order the quantities at the end of the Chapters for these reagents. For example, Tris base is a reagent needed for almost all the Chapters, and the 1 kg size is listed. This does not mean that 1 kg of Tris base is required for each Chapter, rather it means that when we order this reagent, perhaps once/year, we typically order 1 kg. 

Chapter 3 (11-4-99)

Pg. 86:

Procedure

3. Add 0.25 mL of Lowry reagent II and mix well immediately. Note: it is very important to mix well immediately after adding the Lowry reagent II.  Otherwise the Folin reagent becomes less effective.

4. Let stand at room temperature for 30 min.
 
 

Chapter 4 (11-4-99)

Pg. 129:

Because each dodecylsulfate molecule has one negative charge at pH values used for electrophoresis, the net charge of the coated polypeptide chains will be much more negative than that of uncoated chains.
 

Chapter 7 (11-4-99)

Pg. 179: 

1. Hydrate E. coli K-12 cells, 10 mL in 30 mM Tris-HCl plus 0.5 M (20%) sucrose (pH 8.0).

12. Standard (50 micromolar p-nitrophenol (PNP) in 0.2 M Tris-HCl, pH 8.0)
 

Pg. 183:

10. Standard (50 micromolar PNP in 0.2 M Tris-HCl, pH 8.0)

Pg. 186:

22. Dialyze as before (5 mM Tris-HCl, pH 8.0) until the next laboratory period.

MATERIALS

8. Standard (50 micromolar PNP in 0.2 M Tris-HCl, pH 8.0)

10. You do not need HEPES buffer for this experiment.
 

Pg. 194:

METHOD 2: FIXED TIME-ASSAY OF ALKALINE PHOSPHATASE

The basic assumption in this procedure is that the enzyme activity is linear with time up to the point when the reaction is stopped by adding a quenching solution. Advantages of the fixed-time assay are that a large number of samples can be assayed at the same time, the temperature can be controlled by using a water bath, and the spectrophotometric measurements can be done at a convenient pace.

Pg.196:

Clearer instructions for preparation of cells containing alkaline phosphatase. 

We use E. coli EC-1 from Sigma Chemical Co. The cells are freeze-dried. Each student pair will need 0.25 g of freeze-dried cells. In addition, some cells should be used by the instructors to check the protocol,  so that there are samples available for students who make mistakes in the early stages of the protocol. Typically, we save for contingencies and for testing the protocols about 50% of the amount of cells needed for the students in their experiments. 

The freeze-dried cells are suspended in 10 mM Tris-HCl, pH 8.0. We use 1 L of buffer for 25 g of freeze-dried cells. The cells are collected by centrifugation, and resuspended in 1 L of 30 mM Tris-HCl, pH 8.0, 0.5M sucrose. Check the pH, and if it is below pH 8.0 correct it with 1 M Tris base, pH 8.0. Store the cells at 4C for 48 hr before use. 

When checking the protocol, we run the sample up to Stage 1. Then, 20% of the sample is carried through the whole procedure. If students have problems with their preparations, we give them aliquots of the Stage 1 enzyme with which they can carry on. 

Sample calculations: 

Ten student pairs will need 2.5 g cells. 

To check the protocol, you will need 1.25 g cells. 

Of the 1.25 g cells, 80% of the sample (equivalent to enzyme from 1 g of cells) will be held aside as Stage 1 enzyme. Thus, 4/10 of the student groups can mess up and we can provide them with Stage 1 enzyme. The rest of the sample, equivalent to enzyme from 0.25 g of cells, will be used to check the rest of the protocol. 

Pg. 197:

Column buffer: Tris-HCl (5 mM) pH 8.0 containing MgSO4 (5 mM)

Enzyme assay materials (needed for each day of the experiment):
PNPP (1 mM) in Tris-HCl (0.2 M) pH 8.0. (Make 4 liters) (PNPP, Sigma 104-0) Use 371 mg/L.

You do not need the HEPES buffer.
 
 
 

Chapter 8 (10-22-98)

Suggested changes for experiments 8-1 and 8-2. 

Because the Km of Alkaline Phosphatase for PNPP is very low (~8 uM), the experiment as described in the text will not provide the best data. Better results will be obtained if a lower concentration of enzyme is used, such that the reactions proceed more slowly and the linear phase of the reaction lasts longer. We suggest reducing the enzyme concentration to ~15 mU/0.2 mL, instead of using 60 mU/0.2 mL as stated in the text. 

Also, as an independent check of the amount of substrate actually added to each tube, we recommend that the reaction tubes be incubated for at least 30 min after the experiment is completed. Then measure the absorbance to determine how much PNP is produced. It should be the same as the amount of PNPP added. 

Another suggestion is that experiment 8-2 will provide better data if the continuous assay is used instead of the fixed-time assay that is described in the text. Simply set up assays as in experiment 8-1, but include appropriate amounts of phosphate. 

The following is a new experiment.
 

EXPERIMENT 8-3

DETERMINATION OF EFFECT OF TEMPERATURE ON KM AND VMAX OF ALKALINE PHOSPHATASE

OBJECTIVES

The protocol is based on experiment 8-1, but because you cannot control the temperature of the Spectronic 20 sample compartment, you will incubate your samples in appropriate water baths to control the temperature. This requires you to use a fixed time method (see Experiment 8-2) instead of a continuous assay method for determining activity of alkaline phosphatase. You will do the following at four different temperatures. The assay for alkaline phosphatase that you will use, as well as several principles of carrying out enzyme assays are elaborated on pages 191-195.

When using the fixed time method it is important to be sure that the assay is linear (see page 194 about the fixed time assay). Devise a scheme so that you can be sure that the assay is linear before setting up all of the kinetic assays. Hint: Use two different concentrations of enzyme and incubate for different times using a fairly low concentration of substrate.  Remember that the enzyme is likely to have a larger Vmax at higher temperatures than at lower temperatures, so you may want to use a smaller quantity of enzyme for these assays.
 

MATERIALS

1. 1 mM PNPP in 0.2 M Tris-HCl (pH 8.0)

2. 0.2 M Tris-HCl (pH 8.0)

3. Enzyme solution appropriately diluted with Tris buffer (You will determine an appropriate dilution as described above. Probably 15-30 mU in your assay mixtures will be appropriate)

4. 10 N sodium Hydroxide
 

PROCEDURE
 

1.   Determine the appropriate amount of enzyme for the assays.

2. To five 13 x 100 mm test tubes, pipette different volumes of 0.4 mM PNPP so that the amount of PNPP ranges between 30 and 600 nmol.

NOTE: The final concentration of PNPP in 3 mL of reaction mixture should be between 0.01 and 0.2 mM.

3. Add 0.2 M Tris-HCl to make the volume in each tube 2.8 mL. Mix.

4. Incubate the tubes in the selected water bath for 2 min.  Baths will be set up at 25, 35, 45, and 55 °C.

5. Add enzyme solution to each tube (amount determined above).  Strictly time the addition, mix, and place back in the water bath.

6. At the required time (probably 1 or 2 min as determined above for a linear assay), add 0.2 mL of 10 N NaOH to each tube and mix quickly. Be sure that each of the assays has been incubated for the same amount of time when the NaOH is added.

7. Remove the tubes from the water bath, and determine their absorbance at 410 nm.  Use a "no enzyme" blank.

NOTE:  Set the Spec 20 to 100% T with a CLEAN tube before adding the enzyme; this is CRITICAL.  Timing is also CRITICAL, as is wiping the tube CLEAN after removing from the water bath.

8. Determine Km and Vmax for each temperature.  Make some conclusion about the general effects of temperature on alkaline phosphatase activity, such as what is the optimal temperature at which the enzyme functions.  Include in lab report.
 
 

Chapter 9

Under - TESTS TO DESIGN IN DEVELOPING THE ASSAY

Taking the example above, ask whether the LDH will use NADH > 0.01 mM/min (0.062 min (-1) cm (-1) of path.

Pg. 234, bottom

The pH optimum for the kinase activity of CPU is 9.0, while the reverse reaction is optimal at pH 7.5. Although we want to measure the kinase activity, we nevertheless use a suboptimal pH of 8.0 (Tris-HCl).  This is because the coupling enzymes are more effective at pH 8.0.  Be sure that after mixing your reagents you have a final pH of 8.0.

Pg. 237

Under PROTOCOL SUGGESTIONS

We use Tris-HCl, 0.05 M, pH 8.0 for all experiments (Note the change). However, all of the reagents are made up in 50 mM Tris, pH 7.5. Be sure that your measuring buffer (Tris-HCl, 0.05 M, pH 8.0) compensates for the storage buffer.