PMSF binds specifically to the active site serine residue in a serine protease, but does not bind to any other serine residues in the protein. Since PMSF binds covalently to the enzyme at the active serine residue, the complex can be viewed by X-ray crystallography; it can therefore be used as a chemical label to identify an essential active site SER in an enzyme.

Enzyme (active) Ser-O-H + F-SO₂CH₂C₆H₅ ->  EnzymeSer-O-SO₂CH₂C₆H₅ + HF

Serine protease + PMSF -> Irreversible enzyme-PMS complex + HF

PMSF is a cytotoxic chemical that should only be handled inside a fume hood; the LD₅₀ for this compound is less than 500 mg/kg.

Preparation of PMSF (10 mM), 10 ml

1. Weigh out  17.4 mg PMSF
2. Add isopropanol to 10ml and dissolve.
3. No need to filter sterilize.
4. Aliquot and store at –20°C.

PMSF is extremely unstable in aqueous solutions with a half-life of approximately 30 minutes. Add solution immediately before use.

Recommended Suppliers

PMSF: Sigma (P-7626)

The three dimensional structure of ampicillin.

Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. It is often used as a selective agent in molecular biology to select for and to confirm the uptake of genes (e.g., of plasmids) by bacteria (e.g., E. coli). A gene that is to be inserted into a bacterium is coupled to a gene coding for an ampicillin resistance (in E. coli, usually the bla (TEM-1) gene, coding for ?-lactamase). The treated bacteria are then grown in a medium containing ampicillin (typically 50–100 mg/L). Only the bacteria that successfully take up the desired genes become ampicillin resistant, and therefore contain the other desired gene as well. It can be used with Cloaxicillin as well. As a powder ampicillin is white with slight yellow cast and is soluble in water (150 mg/ml).

Preparation  of Ampicillin (100mg/ml), 50ml

1. Weight out 5g Ampicillin
2. Add double distilled H2O to 50ml.
3. Sterilize using a 0.22 µm filter.
4. Aliquot and store at –20°C. (Use at 100µg/ml).

Recommended Suppliers

Ampicillin: Sigma-Aldrich, A-9518

Preparation of acrylamide solution (30%), 500 ml

1. Weigh out 29 grams 2X Acrylamide.
2. Weigh out 1 gram of N,N’-methylenebisacrylamide.
3. Add acrylamide and N,N’-methylenebisacrylamide to 300ml double-distilled H2O.
4. Heat to 37°C to dissolve chemicals.
5. Adjust the final volume to 500ml with double-distilled H2O.

Recommended suppliers

Acrylamide: Serva, 10675

N,N’-methylenebisacrylamide: Serva, 29195

Good’s buffers are twelve buffering agents selected and described by Norman Good and colleagues in 1966. Good selected the buffers based on a number of criteria which make them candidates for use in biochemistry and biological research. Many remain staples in modern biology laboratories.

Selection criteria

Good sought to identify buffering compounds which met several criteria likely to be of value in biological research.

1. pK_a. Because most biological reactions take place at near-neutral pH between 6 and 8, ideal buffers would have pK_a values in this region to provide maximum buffering capacity there.
2. Solubility. For ease in handling and because biological systems are in aqueous systems, good solubility in water was required. Low solubility in nonpolar solvents (fats, oils, and organic solvents) was also considered beneficial, as this would tend to prevent the buffer compound from accumulating in nonpolar compartments in biological systems: cell membranes and other cell compartments.
3. Membrane impermeability. Ideally, a buffer will not readily pass through cell membranes, this will also reduce the accumulation of buffer compound within cells.
4. Minimal salt effects. Highly ionic buffers may cause problems or complications in some biological systems.
5. Well-behaved cation interactions. If the buffers form complexes with cationic ligands, the complexes formed should remain soluble. Ideally, at least some of the buffering compounds will not form complexes.
6. Stability. The buffers should be chemically stable, resisting enzymatic and non-enzymatic degradation.
7. Optical absorbance. Buffers should not absorb visible or ultraviolet light at wavelengths longer than 230 nm so as not to interfere with commonly-used spectrophotometric assays.
8. Ease of preparation. Buffers should be easily prepared and purified from inexpensive materials.

The twelve buffers selected by Good are tabulated below.

A semi-permanent mounting media for immunofluorescence microscopy.

Method

1. Add the following reagents to a 250 ml flask or beaker:

• 24 g analytical grade glycerol (Sigma #G-6279)
• 9.6 g Mowiol 4-88 (Fluka, #81381, can be purchased through Sigma-Aldrich)
• 24 ml distilled water
• 48 ml 0.2M Tris buffer, pH 8.5

2. Stir with a clean stir bar on a hot plate on warm (not boiling)at least 4-5 hours until the majority of the Mowiol powder goes into solution.
3. Aliquot into 50 ml centrifuge tubes, weigh and balance
4. Centrifuge at 5000g for 15 minutes. Carefully remove the supernatant without disturbing the pellet at the bottom of the flask.
5. Aliquot into 15 ml conical tubes – add only 10 mls to each tube to allow for expansion with freezing.
6. Aliquots may be stored at -20C for 12 months. Store at room temperature no more than one month.
7. To use, warm solution to room temperature to eliminate bubble formation. use approximately 10 ul of mounting media for an 18 mm coverslip.
8. Allow slides to dry overnight at room temperature in a light-tight box.

Applications

PBS has many uses because it is isotonic and non-toxic to cells. It can be used to dilute substances. It is used to rinse containers containing cells. PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be ‘dried’ and immobilized to a solid surface. The thin film of water that binds to the substance prevents denaturation or other conformational changes. Carbonate buffers may be used for the same purpose but with less effectiveness. PBS can be used to take a reference spectrum when measuring the protein adsorption in ellipsometry.

Additives can be used to add function. For example, PBS with EDTA is also used to disengage attached and clumped cells. Divalent metals such as zinc, however, cannot be added as this will result in precipitation. For these types of applications, Good’s buffers are recommended.

Preparation

There are many different ways to prepare PBS. Some formulations do not contain potassium, while others contain calcium or magnesium^[1]. One of the most common preparations is described below.

A 10 liter stock of 10x PBS can be prepared by dissolving 800 g NaCl, 20 g KCl, 144 g Na₂HPO₄ · 2H₂O and 24 g KH₂PO₄ in 8 L of distilled water, and topping up to 10 L. The pH is ~6.8, but when diluted to 1x PBS it should change to 7.4. When making buffer solutions, it is good practice to always measure the pH directly using a pH meter. If necessary, pH can be adjusted using hydrochloric acid or sodium hydroxide.

On dilution, the resultant 1x PBS should have a final concentration of 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, and a pH of 7.4.
Another preparation is described in Molecular Cloning by Sambrook, Fritsch and Maniatis, Apendix B.12^[2] as follows:

For 1 litre of 1X PBS, prepare as follows:

1. Start with 800 ml of distilled water:
2. Add 8 g of NaCl.
3. Add 0.2 g of KCl.
4. Add 1.44 g of Na₂HPO₄.
5. Add 0.24 g of KH₂PO₄.
6. Adjust the pH to 7.4 with HCl.
7. Add distilled water to a total volume of 1 liter.

Dispense the solution into aliquots and sterilize them by autoclaving (20 min, 121°C, liquid cycle). Store at room temperature.

References

1. Dulbecco, R. et al. (1954): Plaque formation and isolation of pure lines with poliomyelitis viruses. In: J. Exp. Med. vol. 99 (2), pp. 167-182. PMID 13130792
2. Sambrook, Fritsch, and Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, volume 3, apendix B.12
3. Portions of this article are from “Phosphate buffered saline. In Wikipedia, the free encyclopedia. Retrieved September 17, 2008, from http://en.wikipedia.org/wiki/Phosphate_buffered_saline.” This article has been reviewed for scientific accuracy and is used in accordance with Wikipedia’s GNU Free Documentation License (GFDL).