BME

Molecular BiologyExperiment

SDS-PAGE for protein induction

SKKU BME

3rd grade, 2nd semester

Today Making samples

Boiling the samples

SDS-PAGECoomassie

blue staining

SDS PAGE gel

SDS?

• Sodium dodecyl sulfate, synonymously sodium lauryl sulfate (or laurilsulfate; SDS or SLS, respectively), is a synthetic organic compound with the formula CH3(CH2)11SO4Na. It is an anionic surfactant used in many cleaning and hygiene products. The sodium salt is of an organosulfate class of organics. It consists of a 12-carbon tail attached to a sulfate group, that is, it is the sodium salt of dodecyl hydrogen sulfate, the ester of dodecyl alcohol and sulfuric acid. Its hydrocarbon tail combined with a polar "headgroup" give the compound amphiphilic properties and so make it useful as a detergent.[not verified in body] Also derived as a component of mixtures produced from inexpensive coconut and palm oils, SDS is a common component of many domestic cleaning, personal hygiene and cosmetic, pharmaceutical, and food products, as well as of industrial and commercial cleaning and product formulations.

If no SDS was added?Natïve Polyacrylamide gel electrophoresis

SDS PAGE

Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule.

SDS-PAGE

Materials

Acrylamide gel

Reaction!

TEMED accelerates the rate of formation of free radicals

from persulfate and these in turn catalyze polymerization.

in three steps: chain initiation,

chain propagation, and chain

termination.

Ratio of Acrylamide and Bis-acrylamide

Various pore sizes

Ratio&Resolution

Stacking & Resolving gels• After protein loading…

Stacking & Resolving gels

• After protein loading…

Summary – Imagine the procedure!

What are we doing today?

• Sample preparation

• SDS PAGE gel making

• Load the samples

• Electrophoresis

• Coomassie blue staining

• De-staining

What will you load?

Before and After induction samples…

Ladder Samples…

Sample preparation

• Compare the pellet color of before and after induction samples

• Based on O.D. e.g. Before induction: 0.6 & After induction: 1.2

=> Dilute Before:After = 1:2

• Based on eye-observation

• Dilute with D.W. (50 ul : 100 ul)

• Then, use each 12 ul for sample loading

• 12 ul (each sample) + 3ul (5X SDS buffer)

• Heating at 90C for 5 min

• Keep it in the ice

Before After

Protocol for making gelResolving gel

8ml 12% resolving gel mix

dH2O 2.8ml

30% Acrylamide/Bis-acrylamide 3ml

1.5M Tris, pH 8.8 2.5ml

10% SDS 100ul

150ul 2% APS

15ul TEMED

Stacking gel

4ml 5% stacking gel mix

dH2O 7.3ml

30% Acrylamide/Bis-acrylamide 1.25ml

1.0M Tris, pH 6.8 1.25ml

10% SDS 100ul

50ul 2% APS

5ul TEMED

150 V 1 hr

Assemble the cast

• Glass plate

• White plate

• Spacer

• Cast parts (Red, Gray and black parts)

• Comb

BufferBuffer

120V in stacking

150V in resolving

Check the + and -

Coomassie blue staining

Coomassie blue

• Coomassie Brilliant Blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie Brilliant Blue G-250 differs from Coomassie Brilliant Blue R-250 by the addition of two methyl groups. The name "Coomassie" is a registered trademark of Imperial Chemical Industries.

Bradford assay with CBB• The Bradford protein assay is used to measure the

concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue.

Bradford assay• 1. In acidic condition,

https://www.youtube.com/watch?v=hdb3s4YHkms

Change of color!

What you should do…

• Remove the PAGE gel from the glass plate

• Put the gel in CB staining solution

• Wait for 30 min => Check the blue color

• Put the gel in washing solution for 30 min

• Check the bands

No home work!