There are a number of objectives we will be working on in this and succeeding laboratories. Some of the more important objectives for this time include:
a) Establishing good laboratory note-book habits. (Review the hand out on laboratory note-books.)
b) Establishing good, general laboratory techniques, such as being conscious of laboratory safety, labeling containers, paying attention to details when making observations, reading instructions in advance and knowing what to do and how to do a new technique before attempting it, establishment of a testable null hypothesis, etc.
c) Learning proper use of pipettes and micropipettes.
d) Learning use of the spectrophotometer and cuvettes.
e) Experiencing and coping with the problem of measurement error when trying to establish a valid measurement.
f) Introducing the use of simple statistics to aid in deciding whether or not to reject a null hypothesis.
g) Learning the use of a protein assay, and establish a calibration curve to use when studying cell fractionation in January. (Don't forget what you learn; keep good notes, so that you will be able to do similar measurements at the later time!)
Determine the amount of protein (Bovine Serum Albumin) in three unknown solutions; (Unknown A, Unknown B, and Unknown C).
Determine if the unknown protein solutions differ from each other with respect to their protein concentrations.
Use the Coomassie Blue G-250 Dye method for protein determination. (Several dyes interact with protein, in a yet undefined manner, to yield a change in "color" or light absorbance by the dye-protein conjugate. This change can be quantified with a spectrophotometer. See posted reprints regarding Protein-Dye Assays on the Cell Biology Laboratory web site.
Prepare a calibration curve using known amounts of Bovine Serum Albumin reacted with Coomassie Blue G-250. Plot Absorbency versus amount of protein reacted with Coomassie Blue G-250.
Measure the absorbency resulting from the reaction of Coomassie Blue G-250 with a known volume of the Bovine Serum Albumin unknown.
Determine the amount of protein in the unknown measurement by assuming that equal quantities of protein will result in equal absorbencies when reacted with Coomassie Blue G-250 in the same way; i.e., simply read the protein value from the calibration curve which corresponds to the absorbency obtained after reacting with Coomassie Blue G-250.
After a volume of unknown is found which gives a valid absorbency value, repeat the determination of the unknown several times, to allow use of statistical tests to decide whether the protein unknowns differ from each other.
1. Determine the minimum volume required for using a mini‑cuvette in the spectrophotometer:
2. Obtain ca. 1 ml of Protein Solution (1 mg protein/mL) in a properly labeled 1.5 ml microcentrifuge tube.
3. Use the above Protein Solution to prepare a graded series of proteins for the calibration curve:
Before using the micropipettes, read the instructions for their proper use. Note and assiduously avoid the common first-use mistakes than many students make. Be sure to:
(i) Use a clean micropipette tip. The same tip can be reused if repipetting from the same solution.
(ii) Be sure the micropipette tip is sealed firmly on the end of the pipette.
(iii) Note: bare fingers are loaded with RNAse and oils, even if you just washed them. Keep hour fingers well away from the pipette filling tip, and don’t immerse the part of the tip that you touch into your (clean) solution. Immerse only the tip of the pipette tip into the sample that is being pipetted.
(iv) Fill the pipette slowly. Do not let the filling plunger snap back, or the sample will aerate and adhere onto the upper part of the tip as separate drops which are very hard to transfer. Note: viscous samples take much longer to fill (and discharge).
(v) Check that the tip does not carry an extra drop when you remove the filled tip from your solution. It is a good practice to gently touch the tip to the inside of the container to remove any extra drop.
(vi) Be able to feel the difference in spring pressure between the fill and the blow-out positions of the plunger.
(vii) Discharge the sample slowly. Discharging too quickly will leave part of the sample adhering to the inside of the pipette. This is especially true for viscous samples.
(viii) Be sure that the last part of a drop is transferred. It is a good practice to touch the tip to the inside of the vessel to ensure that the last part of a drop is transferred.
(ix) Be sure you know how to adjust the volume of and read to calibration scales of the variable micropipettes.
(x) Check the calibration of the micropipette by weighing (on a water-resistant tare; on a micro-balance) a measured volume of water. (The density of water at 20oC is 0.9982071 grams/ml. Be aware that the weight of the water drop will change as the water evaporates from the tare.)
(a) Use 1.5 mL microcentrifuge tubes for mixing protein with Coomassie Blue G-250 reagent. Place 0, 10, 20, 40, 60, 80, 100 µg of protein to labeled microcentrifuge tubes. Reuse the pipette tip when measuring from the same solution. Touch the pipette tip to the side of the container to leave or transfer the last drop.
(b) To each test tube add a measured volume of distilled water to make the cuvettes contain the same final volume of solution, which is at least one half of the minimum volume required for the spectrophotometric measurement. Use a clean pipette tip for the distilled water!
Vortex Mixer use:
Prior to using the Vortex Mixer with any noxious or valuable substance, practice using the Vortex Mixer with a similar volume of water. To prevent spills, you must be able to hold the vortexing container above the level of the liquid. If you cannot do this, or if you cannot master the vortex technique, do not mix with the Vortex Mixer!
(c) Mix gently by swirling or with a vortex mixer without frothing. (Frothing denatures proteins; denatured proteins precipitate; and precipitated proteins are not measured! Only soluble protein is accurately measured.)
4. React with Coomassie Blue G250:
(a) Double the volume of solution in the test tube by adding an equal volume of Coomassie Blue G-250 reagent. Check that the pump is set for the correct volume. Be sure to lift the Repipette plunger all the way (gently), before depressing it all the way (gently).
Note: The Protein Dye Solution contains 0.6 N Perchloric Acid. Perchloric Acid is a strong acid. Use safety precautions for acids, including eye-shields!
Note: Newton's Laws of Inertia apply to a moving column of fluid. Operating the pump quickly will result in excess transfer of fluid on both the up-stroke and down-stroke of the pump!
Note: If you end up with differing levels of fluid in your test tubes, something went wrong!
(b) Mix immediately after closing the cover on the microcentrifuge tube by shaking or holding the tube against a vortex mixer (CAUTION, ACID, see use of Vortex Mixer, above).
(c) Allow color to develop for 2 minutes. Measure absorbance using a 1mL cuvette within 10 minutes. (High concentrations of protein‑dye conjugates will eventually precipitate. Precipitated protein-dye conjugate will not me measured accurately. Thus, measure the higher concentrations first.)
5. Prepare and calibrate the Spectrophotometer:
(To increase the stability of the electronic circuits within the spectrophotometer, it should be turned on and allowed to "warm up" for at least 10 minutes prior to making measurements. Do not turn the spectrophotometers off, unless you are sure that you are the last person using it for the day.)
(a) Read the directions for use of the GeneSys 6 Spectrophotometer, especially section 2. You may save data on a floppy disk (page 2-2), move the floppy disk with the data file to the computers set up in the Cell Biology Laboratory SLC 284, and then email the data to yourself as an attached file. Use the "Manual 6" mode for measurements.
(b) Set the spectrophotomet to 620 nm for protein determination using Coomassie Blue G-250. (See page 2-8.)
(c) Transfer an appropriate volume of the reagent "blank" to a cuvette and place in the holder marked "B". (The reagent "blank" has all of materials present in a cuvette except the material to be measured. Use the dye reagent diluted with an equal volume of distilled water.) Be certain the cuvette is turned properly. The outside of the cuvette must be clean (use Kim‑wipes) and free of scratches. (Always use plastic racks for cuvettes.) Close the lid to prevent extraneous room light from being measured.
(d) Press "Measure Blank" to measure the blank.
6. Measurement of absorbencies:
(a) Transfer an appropriate volume of protein-dye to a cuvette and place in an empty position (keep the blank in position "B"). Be sure the solution fills the light path area of cuvette holder. Be sure the cuvette is clean on the outside. Be sure the cuvette is turned properly.
(b) Close the lid and press the dial button according to the number of the cuvette location that is to be measured.
(c) Read the absorbance. Record the absorbance, either directly into your laboratory notebook, or the floppy disk, followed by printing and taping into your notebook.
BE SURE TO RECORD DIRECTLY INTO YOUR LABORATORY NOTE-BOOK:
i. the wavelength of light used,
ii. the slit width (if adjustable and/or known),
iii. the length of the light path through the absorbing substance (cuvette size),
iv. the absorbency of the substance,
v. the substance (if known) and
vi. the concentration of the substance (if known).
BE ESPECIALLY CAREFUL THAT:
i. Dirt is not affecting the pathway of light. (Use clean cuvettes. Clean the outside of the cuvette with clean tissue paper. Centrifuge or filter solutions to remove particulate matter.)
ii. Scratches are not affecting the transmission of light through the cuvette. (Use plastic cuvette racks and do not let the cuvettes rub each other. Do not clean cuvettes with a wire brush; use a plastic brush or a cotton Q-tip.)
iii. The same spectrophotometer is used for related samples. (Or calibrate each spectrophotometer for wavelength selection and for amplifier gain at the wavelengths used.)
(d) Make a graph of Absorbance (ordinate) versus mass of protein (abscissa). Indicate that the points are data points by drawing a small circle around each point.
(e) Calculate by least squares method (or eye‑ball) the best‑fit line in the linear portion of the graph. Use only this range of absorbance for calculating the unknowns.
(f) Performing the calibration curve in triplicate or otherwise increasing the number of data points will increase the precision of your curve. (Remember that for small sample sizes, the median is a better measure of central tendency than the mean. Thus, choose the middle value; do not use averages of three points!)
7. Determine the absorbance of the unknown solutions:
(a) Place a measured volume of unknown protein solution in a labeled 10mm test tube.
(b) Determine the absorbency of these samples using 3.(b)-6.(c), above.
(c) If the absorbance is out of the working range of your calibration curve, then mark the absorbance value as invalid and repeat the measurement using a larger or smaller sample, according to whether the absorbency reading is too low or too high, respectively.
(d) If the absorbance indicated is in the linear (or working) range of the graph and above the lowest detectable amount, then record the valid absorbance value and repeat the determination procedure [3.(b)-6.(c)] at least nine times (for each unknown), to allow calculation of sample variance.
8. Coomassie Blue G250 Scans:
(a) Prepare a blank for the protein assay, with 0 µg of protein.
(b) Prepare a Protein-Coomassie Blue conjugate sample using 50 µg of protein.
(c) Use deionized water for zeroing the spectrophotometer for scanning, in the "B" (blank) holder.
(d) Scan both of Coomassie Blue samples from 400nm to 700nm at 5nm intervals, one at a time, in the 1 or 2 holder positions. (Be sure that the spectrophotometer is set to scan the correct cuvette location!)
9. Clean up:
(a) Remove labels from test tubes and cuvettes
(b) Rinse dirty glassware with water and place in bucket. Cuvettes go into a special bucket. Do not scratch the cuvettes.
10. Write up:
(a) Follow the Protein Determination Report instructions on the 307 Course Syllabus for this lab procedure.
1. Dye solution:
Dissolve 600 mg Coomassie Blue G250 in 50 ml of 95% Ethanol.
Add 750 ml of 0.4 M Perchloric Acid.
Dilute to 1 liter with Distilled Water.
Mix (filter if necessary).
Store at room temperature.
2. Protein stock solution:
Add 500 mg Bovine Serum Albumin (lyophilized) to
500 ml of Distilled Water.
Dissolve without frothing and without heating.
Store at 4oC.
3. Working Protein Solution: (make up just before use)
Dilute 1 part of protein stock solution with
9 parts distilled water, to give 100 µg protein / ml.
Store at 4oC.
4. Unknown protein solutions:
Label three flasks: Protein UNK A, Protein UNK B, Protein UNK C
The unknowns will be prepared by the instructor just prior to use.
5. Spectrophotometers (seven), with micro cuvette holders.
6. Cuvettes 2mm width, 10mm path length.
7. Vortex mixers (four).
8. Repipetters variable , 5ml (two).
9. Plastic test‑tube racks (eight) for cuvettes.
10. Labeling tape with dispensers (two).
11. 12 mm diameter test tubes (ca. 250).
12. Micropipette stations (eight), each with:
micropipette holder and tip bins
micropipette tips for the following micropipettes
100 µl to 1000 µl micropipette
20 µl to 200 µl micropipette
5 µl to 50 µl micropipette
13. Kim‑wipes (seven boxes, by spectrophotometers)
14. Wash bottles, labeled and filled with distilled water (eight).
15. Graduated cylinders, 10 ml (eight).
16. Distilled Water in carboy, labeled.
17. Erlenmeyer flasks, 50 ml (eight).
18. Containers for dirty cuvettes and other glassware, labeled (at least two).
19. Microbalance (one) accurate to 0.1 mg
21. Cotton-tipped, plastic/wooden-handled applicators (30) [or alternatively, plastic brushes (7)] for cleaning cuvettes
22. Parafilm for capping 10mm test tubes during mixing.