Electrophoresis / SDS-PAGE

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PurposeUse gel electrophoresis to

compare protein profiles of fish

What is SDS-PAGE?

SDS (Sodium dodecyl sulphate) is a detergent used to denature proteins and give them a negative charge

PAGE: Polyacrylamide Gel Electrophoresis

It is a technique to separate proteins by their molecular weight

ElectrophoresisThe migration of proteins is affected

by multiple factors involving their structural organization.◦Amino acids can carry either a net

positive, net negative, or neutral charge depending on the combination of amino acids they contain.

To make protein migration rates a function of molecular weight, it is necessary to impose a uniform shape and charge on all of the proteins in the mixture.

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What is so special about SDS?SDS is a negatively charged

detergent.Disrupts secondary and tertiary

protein structures by breaking hydrogen bonds and unfolding protein.

‘Masks’ charge on protein so that all proteins act the same as regards charge.

Prevents protein aggregation.Prevents protein shape from

influencing gel run.

How does SDS-PAGE work?

Proteins (negatively charged due to SDS) move to positive electrode

Proteins separate by sizeSmaller proteins move faster

6

-

+smallest

largest

large

small

Why heat the samples?

Heating the samples helps denature proteins and protein complexes, allowing the separation of individual proteins by size

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s-s

-

+

SDS, heat

proteins with SDS

Why Use Polyacrylamide Gels to Separate Proteins?

Smaller pore size than agarose Proteins much smaller than DNA

◦ average protein = 30-50 kD

◦ “average” DNA = >2000 kD

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Procedure: Day 11. Label one flip-top and one screwcap microtube with the

letter of each fish sample being prepared for electrophoresis.

2. Add 250µl of Laemmli Sample buffer to each labeled flip-top tube.

3. Transfer each fish sample to the appropriately labeled microtube and close the lid.

4. Gently flick the microtubes 15 times with finger to agitate the tissue in the sample buffer.

5. Incubate samples for 5 minutes at room temperature.6. Carefully pour the sample buffer containing the extracted

proteins, but not the solid fish piece, into the correctly labeled screwcap microtube.

7. Heat the fish samples and the actin and myosin standard (AM) for 5 minutes at 95oC to denature the proteins.

8. Store samples in freezer until next class.

Procedure: Day 2

1. Place a yellow sample loading guide on the top of the electrode assembly.

2. Load the samples into the wells following this guide:

Lane Volume Sample

1 empty none

210 µl Kaleidoscope

3 10 µl A

4 10 µl B

5 10 µl C

6 10 µl D

7 10 µl E

8 10 µl F

9 10 µl AM standard

10 empty none

3. Load 10 µl of each protein sample into the designated well.

4. After loading all samples, remove the sample loading guide, place the lid on the tank.

5. Run gel for 30 minutes at a constant voltage of 200V.

6. When the gels are finished running, discard the buffer from the inner chamber, release the cams, and remove the gel cassettes from the assembly.

7. Lay each gel cassette flat on the bench with the short plate facing up. Carefully pry apart the gel plates using your fingertips or a weighing spatula. The gel will adhere to one of the plates.

8. Transfer the plate with the gel to a staining tray containing Bio-Safe Coomassie stain.

9. Stain the gels for 1 hour. Gentle agitation throughout staining time gives best results.

10. After staining for 1 hour, pour off stain and return to bottle.

11. Destain the gels in a large volume of water, changing it several times if possible. Allow gels to destain overnight for best results.