Why is beta-mercaptoethanol used in Western blot?

Why is beta-mercaptoethanol used in Western blot?

All western blot samples have three things: protein extract, cell lysis buffer, and Laemmli (sample) buffer. 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.

Is beta-mercaptoethanol toxic?

BME can be toxic if ingested, and fatal if inhaled or absorbed through the skin. Vapors can irritate the eyes, mucous membranes, and respiratory tract. Symptoms of inhalation exposure may include coughing, sore throat, and/or shortness of breath. BME is combustible as a liquid or vapor!

What is the purpose of the β mercaptoethanol?

The role of beta-mercaptoethanol is to break all the disulfide bonds and denature the protein of interest.

Why is B mercaptoethanol added to the SDS-PAGE?

BME is suitable for reducing protein disulfide bonds prior to polyacrylamide gel electrophoresis and is usually included in a sample buffer for SDS-PAGE at a concentration of 5%. Cleaving intermolecular (between subunits) disulfide bonds allows the subunits of a protein to separate independently on SDS-PAGE.

How do you neutralize beta mercaptoethanol?

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.

How do you take dithiothreitol?

General Protocol to Reducing Cysteines in a Protein or Peptide Solution

  1. Make a 1M dithiothreitol DTT stock solution in water, best to make fresh (try not to inhale the DTT).
  2. Add DTT to the protein or peptide solution(which is in a buffer) to a final concentration of 1mM to 10mM DTT. Incubate for 10 min. to 30 min.

What does 2-mercaptoethanol do in SDS-PAGE?

2-Mercaptoethanol is used to reduce disulfide linkages in solubilizing proteins for gel electrophoresis (typically used in SDS-PAGE sample buffer at 5% concentration). Cleaving intermolecular (between subunits) disulfide bonds allows the subunits of a protein to separate independently on SDS-PAGE.

How do you dilute B mercaptoethanol?

Add 1.7 µl (0.8 µl for JM109) of beta-mercaptoethanol provided with the kit or a fresh 1:10 dilution (stock 14.4 M) of beta-mercaptoethanol (diluted in high-quality water) to the 100 µl of bacteria, giving a final concentration of 25 mM. Swirl the contents of the tube gently.

How long is beta-mercaptoethanol good for?

1 month
Beta-Mercaptoethanol (ß-ME) is stable for 1 month, but Buffer RLT itself is stable for at least 9 months at room temperature (15 to 25°C).