1.Purpose:

To describe the steps necessary to perform an SDS-PAGE analysis of a protein sample

2.Scope:

This SOP covers the preparation of SDS-PAGE protein gels suitable for Coomassie staining or Western Blotting.

3.Responsibilities:

1. It is the responsibility of the Supervisor to ensure that this SOP is performed as described and to update the procedure when necessary.

2. It is the responsibility of the Analyst to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

4.References:

1. XCell SureLock® Mini-Cell User Guide, Publication Part number IM-9003

2. GelCode Blue Stain Reagent Instructions, Thermo Scientific

5.Precautions:

1. Routine care should be exercised in the handling of buffers and samples of biological materials, which may have harmful biological activity in the case of accidental ingestion, needle stick etc.

2. Gloves, a lab coat and protective eyewear should be worn when handling buffers and samples.

3. Always wear gloves when handling polyacrylamide gels.

6.Materials:

1. 4-20% Tris-Glycine Gel, Invitrogen Novex WedgeWell (Reference # XP04200BOX)

2. 10X Tris/Glycine/SDS Running Buffer, BIO-RAD Catalog # 161-0732

3. GelCode Blue Stain Reagent, Thermo Scientific (Product # 24590)

4. XCell SureLock Gel Box

5. Gel Knife

6. Power Supply

7. Heating block set at 95ºC

8. 1.5 ml microfuge tubes

9. 2X Laemmli Sample buffer, BIO-RAD catalog #161-0737 with added ß mercaptoethanol

10. Precision Plus Protein Kaleidoscope Ladder, (BioRad catalog # 161-0375)

11. Gel loading pipette tips

12. 10 ml syringe and 21G2 needle

13. Ice bucket and ice

7.Procedure:

1. Prepare Samples.

Note: protein samples should be kept on ice while preparing the samples.

1. Fill the needed number of holes in the heating block with Milli Q water.

2. Turn the heat block on and set the temperature to 95°C to preheat.

3. Label one microfuge tube for each sample.

4. For each sample to be analyzed;

   1. Determine the amount of total protein to be loaded on the gel.

   2. Using the sample total protein concentration, calculate the sample volume equal to the amount of protein to be loaded.

   3. Calculate the volume of Milli Q water needed to add to the sample volume to bring the combined water + sample volume to 15µl.