2-Mercaptoethanol is used to reduce disulfide linkages in solubilizing proteins for gel electrophoresis (typically used in SDS-PAGE sample buffer at 5% concentration). Also it reduces excess oxidative polymerization of catalysts.

Denaturing ribonucleases Numerous disulfide bonds make ribonucleases very stable enzymes, so 2-mercaptoethanol is used to reduce these disulfide bonds and irreversibly denature the proteins. This prevents them from digesting the RNA during its extraction procedure.

SDS imparts uniform negative charge and linearises your protein and Beta-mercaptoethanol breaks cysteine-cysteine disulphide bridges. Heating your protein containing SDS and Beta-mercaptoethanol helps denature the protein. Heating speeds up this breakdown process and the amount of heating is to be optimized in the lab.

Addition of β-Mercaptoethanol (β-ME) to a final concentration of 24 mM has been shown to increase the transformation efficiency of NEB 5-alpha by 140%. The effect on transformation efficiency may be different when using plasmids other than pUC19. Add 0.8 ìl of 1.5 M β-ME to 50 ìl of cells.

Beta-mercaptoethanol (BME) is a reducing agent that acts on disulfide bonds; in the absence of BME, proteins with disulfide bonds retain some shape and do not electrophorese consummately by molecular weight.

Description. Gibco™ 2-Mercaptoethanol (also known as beta-mercaptoethanol or BME) is a potent reducing agent used in cell culture media to prevent toxic levels of oxygen radicals.

Beta-Mercaptoethanol is considered a “severe” poison, causing “irritation to the nasal passageways and respiratory tract upon inhalation, vomiting and stomach pain through ingestion, and potentially fatal absorption if it contacts the skin”.

B-ME is a reducing agent used to eliminate ribonucleases released during cell lysis so that it doesn’t digest the RNA during the isolation.

β-mercaptoethanol molarity Sigma-Aldrich provide BME concentration 14.3 M (pure liquid), yet the openwetware.org protocol for SDS sample buffers gives a molarity of 14.7 M.

BME odor can be neutralized using standard household bleach. Bleach acts as an oxidizer and converts the thiol group of beta mercaptoethanol into a sulfonic acid derivative which eliminates the natural gas odor. Be sure to absorb any excess BME liquid with an inert absorbent prior to odor decontamination with bleach.

Gibco® 2-Mercaptoethanol (also known as beta-mercaptoethanol or BME) is a potent reducing agent used in cell culture medium to prevent toxic levels of oxygen radicals. Mercaptoethanol is not stable in solution so most protocols require daily supplementation.

Since BME is fairly volatile, EH&S surmised that the lingering odor was related to a small amount that had vaporized; however, the odor was also present down the hallway, approximately 30 feet from the lab.

Transportation and Storage: Do not store near sources of ignition, oxidizing agents, acids, alkaline compounds, or any other incompatible materials. Store BME in a well-ventilated area. Transport toxic liquids in secondary containment, preferably a polyethylene or other non-reactive acid/solvent bottle carrier.

Storage and Special Handling Store in a tightly closed container in a cool, dry area that is well ventilated, such as a vented cabinet. Do not store on shelf. Store in a secondary container by itself. Compounds incompatible with Beta Mercaptoethanol are oxidizers, acids, metals, and ignition sources.

Laemmli sample buffer is especially formulated for protein sample preparation to be used in the Laemmli SDS-PAGE system. A protein sample is mixed with the 2X sample buffer (1:1) and heated in boiling water for 2-5 min. The 2-mercaptoethanol reduces the intra and inter-molecular disulfide bonds.

5%

Store BME in a sealed secondary container in a well-ventilated area. The container must be tightly closed, resealed, and stored upright to avoid leakage. Avoid storing on the floor. Transport toxic liquids in secondary containment, preferably a polyethylene or other non-reactive acid/solvent bottle carrier.

Substance is neither a known nor an anticipated carcinogen. Not listed by NTP, IARC, or OSHA. Toxic effects possible by inhalation, ingestion, and skin absorption.

Store at Room Temperature (15-30 °C).

SDS-PAGE of proteins that have been reduced with mercaptoethanol is useful for measuring the monomer molecular weight. Reduction of the disulfide bonds is important for allowing the protein to become completely unfolded so that it migrates properly for its molecular weight.

For your notebook, a common and easy to make recipe for a 2X concentrated Laemmli buffer is: 4% SDS, 10% beta-mercaeptoethanol, 20% glycerol, 0.1? Tris pH 6.8, and 0.005% of bromophenol blue. A concentrated Laemmli buffer can be stored at 4oC for at least a year without worrying about its effectiveness.

dithiothreitol (DTT or Cleland’s reagent) may be used at a final concentration of 350 mM (54 mg/ml). Dilute 1 part sample with 1 part Laemmli sample buffer. More sample buffer can be added if necessary.

The amount of 4X SDS Loading buffer I would add is 1/4 of the total well volume (30 μL): 30μL/4 = 7.5 μL 4X SDS Loading Buffer. So far: 10 μL protein + 7.5 μL loading buffer = 17.5 μL.

DTT quantitatively reduces disulfide bonds and maintains monothiols in a reduced state (see Reference 1). At a final 0.1 M concentration, DTT is also widely used for disruption of protein disulfide bonds in SDS-polyacrylamide gel electrophoresis.

3 years