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SDS Protocol

This document provides instructions for preparing stock solutions and performing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). It lists the components and concentrations needed to make the resolving gel and stacking gel. The procedure involves assembling the apparatus, pouring the resolving gel and allowing it to solidify, then pouring the stacking gel and adding the comb to form wells. The samples are then loaded into the wells for electrophoresis. Safety precautions are noted for certain chemicals.

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109 views3 pages

SDS Protocol

This document provides instructions for preparing stock solutions and performing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). It lists the components and concentrations needed to make the resolving gel and stacking gel. The procedure involves assembling the apparatus, pouring the resolving gel and allowing it to solidify, then pouring the stacking gel and adding the comb to form wells. The samples are then loaded into the wells for electrophoresis. Safety precautions are noted for certain chemicals.

Uploaded by

Saif
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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SDS-PAGE Protocol

Stock solutions

1. Polyacrylamide (PAA 30 %) 29:1

Weigh 1 gram of N, N′-Methylenebisacrylamide and 29 g of acrylamide in a 100 ml flask

and dissolve in 50 ml of distilled water. Make to the volume. Store at 4 C.

2. Tris Base 1.5 M (pH 8.00)

Weigh 18.15 g of Tris Base in a 100 ml flask. Dissolve in 50 ml distilled water. Adjust

pH to 8.80 with 1 N HCL. Bring to the volume with distilled water. Store either at room

temperature or 4 C.

3. Tris HCL 0.5 M

Weigh 3 g of tris HCL in a 50 ml flask, and dissolve in 20 ml distilled water. Adjust pH

to 6.8 with NaOH pellets and bring to the volume with distilled water. Store either at

room temperature or 4 C.

4. Sodium dodecyl sulfate (SDS) 10 %

Dissolve 10 g of SDS in 50 ml distilled water. After it is dissolved completely, make final

volume to 100 ml with distilled water. Store at room temperature.

5. Ammonium persulfate (10 %)

Weigh 10 g of ammonium persulfate in 10 ml amber flask, dissolve in 5 ml distilled

water. Bring to the volume with distilled water. Store at 4 C.

6. TEMED

No need for preparation. Is used directly. Store either at room temperature or 4 C.


Procedure

To make the resolving gel, pipette the following solutions:

Solution Volume

PAA 30 % 6 ml

Tris Base pH 8.80 3.75 ml

Distilled water 4.95 ml

TEMED 20 µl

APS 10 % 250 µl

SDS 10 % 150 µl

Stacking Gel

Solution Volume

PAA 30 % 2.5 ml

Tris HCL pH 6.80 3.75 ml

Distilled water 8.45 ml

TEMED 20 µl

APS 10 % 250 µl

SDS 10 % 150 µl

First assemble the apparatus.

Pour the resolving gel and pipette 100-250 µl of propanol on it.

After it solidifies, wash the propanol with distilled water and use filter paper to remove

any excess of the distilled water.


Pour the stacking gel then and place the comb into it.

The space between resolving gel and the comb should be 1-2 cm.

After it solidifies, carefully remove the comb. If there are bubbles in the wells, wash them

out with SDS running buffer. Pipette the samples into the wells.

Note

Add TEMED and APS at the very last i.e. when you are ready to pour the gels in between the

glass plates.

SDS is toxic in powdered form so face masks should be used.

Acrylamide is a neurotoxin so should be used very carefully.

TEMED should be added in a fume hood as it has an unpleasant smell and is toxic as well.

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