Laemmli Buffer: SDS-PAGE Sample Loading Buffer 2X (Reducing)

Overview

Assay Principle

Using bromophenol blue dye, SDS-PAGE Protein Loading Buffer is a ready-to-use 2X solution. It contains 4% SDS, 20% glycerol, 200mM DTT, 0.01% bromophenol blue and 0.1 M Tris HCl. It can be used for SDS-PAGE protein loading of conventional proteins. It is especially formulated for protein sample preparation to be used in the Laemmli SDS-PAGE system. It is sufficient to prepare 12 mL protein samples. And the whole sample preparation process can be finished within 5 minutes.

Assay Protocol

1. Equilibrate SDS-PAGE Protein Loading Buffer 2X to room temperature or thaw Loading Buffer in a water bath no higher than 30°C.

2. Mix one volume of SDS-PAGE Protein Loading Buffer 2X with one volume of protein sample (i.e. add 1mL protein sample into 1 mL Loading Buffer).

3. Boil sample for 3-5 min.
Note: If semitransparent viscous substance remains after boiling, boil sample for another 5-10 min or add 1X SDS-PAGE Protein Loading Buffer which is diluted from 2X SDS-PAGE Protein Loading Buffer to the sample and then boil for another 3-5 min.

4. Allow sample to cool to room temperature.

5. Load sample into the wells of the SDS-PAGE gel and begin electrophoresis.

6. Stop electrophoresis when bromophenol blue dye front reaches to the bottom of the gel.

General Notes

1. SDS-PAGE Protein Loading Buffer 2X contains DTT which has a slightly irritating odor.

2. SDS-PAGE Protein Loading Buffer 2X should be completely dissolved before use.

3. Please wear the lab coat and disposable gloves and operate in a laboratory hood.

Validation Images & Assay Conditions

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