Electrophoresis and VisualizationElectrophoresis and Visualization

SDS-Page Part IISDS-Page Part II

Boiling LysatesBoiling Lysates Resultant proteins take on a rod-like

shape and a uniform negative charge-to-mass ratio proportional to their molecular weight

Resultant proteins take on a rod-like shape and a uniform negative charge-to-mass ratio proportional to their molecular weight

Proteins are being denatured by the combination of SDS and heat

ElectrophoresisElectrophoresis Use polyacrylamide gel

Stacking gel- Large pore size and stacks proteins Separating gel- Small pore size and Separates

proteins base on molecular weight Why acrylamide?

Acrylamide gel: tight matrix that is ideal for protein separation

Smaller pore size than agarose Proteins are much smaller than intact

chromosomal DNA

Use polyacrylamide gel Stacking gel- Large pore size and stacks proteins Separating gel- Small pore size and Separates

proteins base on molecular weight Why acrylamide?

Acrylamide gel: tight matrix that is ideal for protein separation

Smaller pore size than agarose Proteins are much smaller than intact

chromosomal DNA

How does an SDS-PAGE gel work?

How does an SDS-PAGE gel work?

Negatively charged proteins move to the positive electrode

Smaller proteins move faster

Proteins separate by size

Negatively charged proteins move to the positive electrode

Smaller proteins move faster

Proteins separate by size

-

+

s-s

SDS, heat

proteins with SDS

The SDS-PAGE apparatusThe SDS-PAGE apparatus

Tracking the samplesTracking the samples You will monitor the

progress of electrophoresis to ensure your samples don’t run off the gel Using

Bromophenol Blue that is in the sample buffer

Refer to next slide

You will monitor the progress of electrophoresis to ensure your samples don’t run off the gel Using

Bromophenol Blue that is in the sample buffer

Refer to next slide

What is in the sample Buffer?*Tris buffer to provide appropriate pH*SDS (sodium dodecyl sulphate) detergent to dissolve proteins and give them a negative charge*Glycerol to make samples sink into wells*Bromophenol Blue dye to visualize samples

What is in the sample Buffer?*Tris buffer to provide appropriate pH*SDS (sodium dodecyl sulphate) detergent to dissolve proteins and give them a negative charge*Glycerol to make samples sink into wells*Bromophenol Blue dye to visualize samples

VisualizingVisualizing When electrophoresis is complete,

you will stain with Coomassie Blue and Destain The stain and destain contains

methanol and acetic acid that helps to fix the proteins in the gel.

Coomassie Blue binds to proteins

When electrophoresis is complete, you will stain with Coomassie Blue and Destain The stain and destain contains

methanol and acetic acid that helps to fix the proteins in the gel.

Coomassie Blue binds to proteins

What next?What next?Obtain information about the markerObtain information about the marker

Obtain a picture of your gelObtain a picture of your gel

Calculate Rf Value and GraphCalculate Rf Value and Graph

Dye front

Calculating Rf valuesCalculating Rf values Calculating Rf values

Rf = distance migrated by the moleculedistance migrated by the dye front

Plot a standard curve on a log graph paper or regular graph paper. You must convert to log10(MW of proteins in the

marker if you use regular graph paper) Use your curve in the identifying the unknown and

answer the questions in your lab manual

Calculating Rf values

Rf = distance migrated by the moleculedistance migrated by the dye front

Plot a standard curve on a log graph paper or regular graph paper. You must convert to log10(MW of proteins in the

marker if you use regular graph paper) Use your curve in the identifying the unknown and

answer the questions in your lab manual

kDa Rf203 8.5 135 12.0

86 18.5

19 41.5

33 34.0

8 44.5

41 28.0

CautionCaution Sample buffer waste- contains a

hazardous reducing agent, Beta mercaptoethanol -stinks like rotten eggs Eppendorf tubes should be thrown

into the sample buffer waste container

Sample buffer waste- contains a hazardous reducing agent, Beta mercaptoethanol -stinks like rotten eggs Eppendorf tubes should be thrown

into the sample buffer waste container

Lab summaryLab summary Don’t forget to include the protocol

from Part I! Answer all the questions Make sure to include tables (of both

the standard measurements and unknown measurements) and your graph!

Conclusions?

Don’t forget to include the protocol from Part I!

Answer all the questions Make sure to include tables (of both

the standard measurements and unknown measurements) and your graph!

Conclusions?