Crystallography‎ > ‎Protocols‎ > ‎


TCEP [Tris(2-carboxyethyl)phosphine)]

A reducing agent, prepared and used as a hydrochloride salt (TCEP-HCl) with a molecular weight of 286.65 gram/mol, soluble in water, used as a reducing agent to break disulfide bonds within and between proteins. Other two most common agents used for the same purpose: DTT and β-mercaptoethanol. TCEP has the advantages of being odorless, a more powerful reducing agent, an irreversible reducing agent, more hydrophilic, and more resistant to oxidation in air.

BME (2-mercaptoethanol)

Is the chemical compound with the formula HOCH2CH2SH. It is a hybrid of ethylene glycol and ethanedithiol. ME, as it is commonly abbreviated, is used to reduce disulfide bonds and can act as a biological antioxidant as it scavages hydroxyl radicals (amongst others) . It is widely used because the hydroxyl group confers solubility in water and lowers the volatility, and hence odour, of the thiol.


Inhibits translation by blocking peptidyl transferase on the 70S ribosomal subunit; It inhibits protein synthesis and blocks host DNA synthesis but has no effect on replication of relaxed plasmids.
For preparing 10 mg/ml stock solution:
Measure 100 mg of Chloramphenicol (MW323.1) into a Falcon tube; add 100% EtOH to 10 ml. Store at -20.

Storage: dessicator at room temperature
Ordering info: Sigma C-0378


Dimethyl sulfoxide (DMSO) is a chemical compound with formula (CH3)2SO.
Is a solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water.

DTT (dithiothreitol)

DTT is the common name for a small-molecule redox reagent known as Cleland's reagent. DTT's formula is C4H10O2S2; its oxidized form is a disulfide-bonded 6-membered ring. Its name derives from the four-carbon sugar, threose. DTT is an unusually strong reducing agent, owing to its high conformational propensity to form a six-membered ring with an internal disulfide bond.

For making10 ml of 1M stock solution
Dissolve 1.55 g of DTT in 8 ml of H2O and bring the volume up to 10 ml with ddH2O. Aliquot solution into microtubes with 1 ml per tube. Store in -20¡C.
Storage: DTT powder is stored in dessicator at -20¡C
Ordering info: GibcoBRL15508-013

EDTA (ethylenediamine tetraacetic acid)

EDTA is the chemical compound ethylenediaminetetraacetic acid. EDTA is a chelating agent, forming coordination compounds with most monovalent, divalent, trivalent and tetravalent metal ions, such as silver (Ag+), calcium (Ca2+), manganese (Mn2+), copper (Cu2+), iron (Fe3+) and zirconium (Zr4+). EDTA contains 4 carboxylic acid and 2 tertiary amine groups that can participate in acid-base reactions. EDTA forms especially strong complexes with Mn, Cu, Fe(III), and Co(III).
Used in scavenging metal ions: in biochemistry and molecular biology, ion depletion is commonly used to inactivate enzymes which could damage DNA or proteins.

Guanidine-HCl/Guanidinium hydrochloride

Chaotropes, substances that disrupt the structure of water interactions, help to solubilize hydrophobic proteins and peptides.
In addition to increasing solubility of hydrophobic molecules, guanidine is a general protein denaturant, unfolding proteins and altering their three-dimensional structure.Some proteins will be irreversibly altered upon interaction with guanidine solutions.

Molecular formula: CN3H5•HCl
Molecular weight: 95.54


Isopropyl β-D-1-thiogalactopyranoside; used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of thelac operon. Unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from "eating up" or degrading the inductant; therefore the IPTG concentration remains constant. IPTG induces the transcription of the gene coding for beta-galactosidase, an enzyme that promotes lactose utilization, by binding and inhibiting the LacI repressor. In cloning experiments, the lacZ gene is replaced with the gene of interest and IPTG is then used to induce gene expression. Many regulatory elements of the lac operon are used in inducible recombinant protein systems; IPTG is an effective inducer in the concentration range of 100 μM to 1.5 mM.

PMSF (phenylmethylsulfonyl fluoride)

PMSF is to inhibit serine proteases, e.g., chymotrypsin, thrombin, and papin. It is inactivated in aqueous solutions. The rate of inactivation increases with increasing pH and is faster at 25¡C than at 4¡C. e.g. the half-life of a 20mM aqueous solution of PMSF is ~35 min at pH8.0.
For making 10 mls:
Dissolve 0.174 g of PMSF (MW 174.20) in 10 ml of 100% EtOH (or 100% Isopropanol). Keep cap tightly sealed and store at -20 C. It might only be good for 1 week.
Storage: dessicator in room temperature
Ordering Info: Sigma P-7626
Reference: Sambrook and Russell. Molecular Cloning, A Laboratory Manual 3rd Edition

Making 0.5 M stock:
For 1000 ml, add 186.1 g of Na2EDTA2H2O (MW 372.24) to 800 ml of ddH2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (~20 of NaOH pellets). Bring the volume up to 1000 ml with ddH2O. autoclave. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to 8.0 by the addition ofNaOH.
Dilute to 0.1 M of EDTA:
Do 1:5 dilution with ddH2O. i.e. add 400 ml of ddH2O into 100 ml of 0.5 M EDTA
Storage: Chemical Cabinet
Ordering info: GibcoBRL 15576-028

SDS (sodium dodecyl sulfate)

Sodium dodecyl sulfate (SDS or NaDS) (C12H25NaO4S), also known as sodium lauryl sulfate (SLS), is an ionic surfactant. The molecule has a tail of 12 carbon atoms, attached to a sulfate group, giving the molecule the amphiphilic properties required of a detergent. SDS is commonly used in preparing proteins for polyacrylamide gel electrophoresis (SDS-PAGE). SDS works by disrupting non-covalent bonds in the proteins, thereby denaturing them, causing the molecules to lose their native shape (conformation). Also, anions of SDS bind to the main peptide chain at a ratio of one SDS anion for every two amino acid residues. This effectively imparts a negative charge on the protein that is proportional to the mass of that protein (about 1.4 g SDS/g protein). This new negative charge is significantly greater than the original charge of that protein. The electrostatic repulsion that is created by binding of SDS causes proteins to unfold into a rod-like shape thereby eliminating differences in shape as a factor for separation in the gel.

Making 1000 ml of 20% SDS stock ( Wear mask and weigh SDS in the hood):
Dissolve 200g of SDS (MW 288.38) in 900 ml of H2O. Heat to 68¡C and stir with a magnetic stirrer to assist dissolution, then adjust the volume to 1000 ml with ddH2O. Store at room temperature. Do not autoclave.
Dilute to 2% SDS:
Do 1:10 dilution with ddH2O. e.g. add 100 ml of 20% SDS into 900 ml of ddH2O.
Storage: chemical cabinet
Ordering info: GibocoBRL 15525-017