Spectrophotometry & Dilutions
Microbiology 542 -- Eric Martz
Goals: I. Optimal Dilutions.

Introduction/Rationale. Reagent dilutions are needed daily in biochemical or immunological lab work. It is important to know how to design and perform dilutions which are accurate and which meet the needs of the situation.

Single Dilution Exercise. You are provided with a concentrated stock solution of a yellow solute (p-nitrophenol), labeled "NP Stock". Make a dilution which reduces the concentration by 37-fold, which is a 1/37 dilution.

  1. What do you need to know? Before making your dilution, make a list of things you need to know, and check it with your instructors. (Suppose you are in a research lab and you need to make a dilution of a concentrated reagent stored in the refrigerator. What do you need to know in order to make a dilution which will meet the needs of the situation?)

  2. Record the serial numbers of the pipeters you use (if they have serial numbers); else label the pipeters with tape and marker. (Why is this important?)

  3. Each student (not each pair) should make the 1/37 dilution four times, identically, to test reproducibility. Put 200 microliters of each of your four dilutions, in duplicate, in column 2 (first student) or column 8 (second student) of your 96-well plate (total 8 wells/student)

II. Serial Dilutions.

  1. Inspect your 96-well plate. Rinse top and bottom if it is dirty. Be careful not to scratch the bottom with a paper towel -- scratches increase absorbance! Flip rinse water vigorously into the sink (ask an instructor to demonstrate); if done properly, this leaves no significant volume in the wells.
  2. Select one of your four 1/37 dilutions. In the 96-well plate, make three serial dilutions as follows in column 3 (first student) or 9 (second student). Into wells A, B, C put 0.1 ml diluent. Add 0.1 ml of the 1/37 dilution to well A and mix by pipeting in and out. Remove 0.1 ml from well A and add it to well B; mix. Similarly transfer 0.1 ml from B to C, mixing. Remove 0.1 ml from well C and discard it so all four wells contain the same total volume.

  3. Record the serial numbers of the pipeters you use (if they have serial numbers); else label the pipeters with tape and marker. (Why is this important?)

  4. Repeat the serial dilutions three times, so each student now has four replicate series of dilutions (first student in columns 3, 4, 5, 6; second student in columns 9, 10, 11, 12).

  5. Put 0.2 ml of diluent in well A1, which the reader assumes to be the blank.

  6. After you have completed parts I and II, take your plate to one of the 96-well plate readers and read the entire plate twice at wavelength 405 nanometers.

Data analysis & comprehension questions.

  1. Does it matter whether you discard 0.1 ml from well C after mixing? Why? After all, the concentration stays the same either way.
  2. Is 405-nanometer light visible? What color is it? Why is 405 nm suitable for measuring absorbance of NP (which is yellow)?
  3. From one reading, determine the mean, SD, and SEM for all 4 of your 1/37 dilutions. Is this mean significantly different from that of your lab partner?
  4. Write the mean, SD, SEM from the previous step on the blackboard. When at least 4 students have written their results on the blackboard, find the highest and lowest. Are they significantly different? If so, what are the possible explanations for the discrepancy?
  5. From one reading, determine the mean, SD, and SEM for the four replicates of the last (row C) of your serial dilutions. How does the SD for this compare with the SD for your eight 1/37 dilutions? How do you interpret this?
  6. Is the mean for the last row of your serial dilutions significantly different from that of your lab partner?
  7. How close is the mean to what what it should be, based on the dilutions you performed? How do you interpret any discrepancy?
  8. From your two readings, determine the mean, SD, and SEM for two pairs of readings of the same wells of your serial dilutions in rows C. For example (for first student) 
      C2 first reading, C2 second reading
  C3 first reading, C3 second reading
  9. Which contributes more variation to your readings: the 96-well plate reader, or pipetting?
  10. How does the precision of your serial dilutions compare with the precision of which the pipet is capable, according to the manufacturer?
  11. How can you most easily determine the accuracies of deliveries from a P200 or a P1000?

III. Protein Concentration from Absorbance at 280 nm.

Aromatic amino acids, notably tryptophan, tyrosine, and phenylalanine, absorb light maximally at 280 nanometers. DNA and RNA bases absorb maximally at 260 nanometers. A protein solution which has no contamination with nucleic acids will have an absorbance ratio, A260/A280, of less than 0.6. A substantially larger value is consistent with nucleic acid contamination. If uncontaminated, the concentration of protein in aqueous solution can be determined from the absorbance at 280 nanometers, provided the extinction coefficient is known for the protein in question, since the amounts of aromatic amino acids per milligram of protein varies among different proteins.

Protein Human Rabbit Other
IgG 13.6 13.5 -
Secretory IgA 12.6 13.5 -
IgM 11.8 - -
IgE 15.3 - -
Fab - 15.0 -
Fc - 12.0 -
J chain 6.8 - -
Bovine Serum Albumin - - 6.7
This table of protein extinction coefficients is copied from Immunochemistry in practice by Alan Johnstone and Robin Thorpe, Blackwell, 1982. The extinction coefficients are for a 1% solution at 280 nm.

 

 

 

  Unknown Determination.

  1. Each pair: Fill three cuvettes with: blank buffer, unknown IgG, unknown BSA.
  2. Each student: Read the A280's. Part of this exercise is to learn to set up the spectrophotometer correctly; therefore, an instructor will misadjust the specrophotometer so you can have the experience of adjusting it correctly. All spectrophotometers need attention to these steps:
    1. Select correct light source for UV or visible light.
    2. Adjust wavelength.
    3. Adjust so that blank reads zero absorbance.
    4. Read your sample(s).
    5. Re-read your blank to see how far the instrument has drifted.
  3. Calculate mg/ml for each protein unknown.
  4. Pure monoclonal antibodies typically half-saturate antigen (15 min, room temperature) at 2 nanomolar. Assuming 150,000 kD for IgG, how would you prepare 10 ml IgG at 2 nanomolar from the "IgG unknown" stock, using the concentration you determined in the previous step?
  5. What simple tests could you do to verify that a spectrophotometer is performing correctly, and is being correctly used? How about verification of an analytical weighing scale (range up to 50 grams with reading to 0.1 milligram)? How about verification of a flow cytometer?
  6. What are the pros and cons of using a spectrophotometer vs. an 96-well plate absorbance reader.
  7. Any of the above questions may be on a forthcoming quiz.

Before leaving.

  1. Have an instructor check your results and conclusions.

  2. Rinse your 96-well plate with distilled water and give it to the instructors to store between classes.

  3. Rinse your cuvettes with distilled water and give them to the instructors.