SDS-PAGE Protein Loading Buffer 5X (Reducing)

General Description

Boster’s SDS PAGE Sample Buffer 5X (Reducing) is the most commonly used sample buffer for Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) of denatured proteins in the Laemmli SDS-PAGE system.

This buffer is very important in the preparation of protein samples and loading them onto a gel. Boster protein loading buffer is a 5X reducing hence it requires less sample dilution but yet accommodating more proteins load in a well.

Overview

Assay Principle

Using bromophenol blue dye, SDS-PAGE Protein Loading Buffer is a ready-to-use 5X solution. It contains 10% SDS, 500Mm DTT, 50% Glycerol, 500mM Tris-HCL and 0.05% bromophenol blue dye. 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 15 mL protein samples. The whole sample preparation process can be finished within 5 minutes.

Remember to avoid common mistakes that may occur such as underloading or overloading of proteins. This can be avoided by considering the amount of protein available using protein assay. Loading less protein may lead to poor detection of the bands while on the other hand overloading the protein would lead to the formation of streaks or even smears due to protein aggregation.

It is always advisable to run you gel electrophoresis markers together with the protein samples so that you can observe accurate protein sizes and also act as control

Assay Protocol

1. Equilibrate SDS-PAGE Protein Loading Buffer 5X 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 5X with four volume of protein sample (i.e. add 4mL 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 5X 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 5X contains DTT which has a slightly irritating odor.

2. SDS-PAGE Protein Loading Buffer 5X contains DTT which has a slightly irritating odor.

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

Validation Images & Assay Conditions

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