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SDS-PAGE Gel Preparation Protocol

This document provides experimental procedures for performing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). It describes preparing a 7.5% acrylamide separation gel and stacking gel, casting the gels, loading protein samples mixed with sample buffer, running the gel electrophoresis at 130V, and then staining or blotting the gel. Safety precautions are noted for acrylamide which is neurotoxic. The protocol is based on a 1970 paper by Laemmli describing the technique.

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154 views2 pages

SDS-PAGE Gel Preparation Protocol

This document provides experimental procedures for performing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). It describes preparing a 7.5% acrylamide separation gel and stacking gel, casting the gels, loading protein samples mixed with sample buffer, running the gel electrophoresis at 130V, and then staining or blotting the gel. Safety precautions are noted for acrylamide which is neurotoxic. The protocol is based on a 1970 paper by Laemmli describing the technique.

Uploaded by

jordanleila
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd

Category: Experimental Procedures

SDS-PAGE standard Author: Admin eLabJournal

Labels: Western blotting

Materials
- Separation buffer (4X)
- Stacking buffer (4X)
- 30% acrylamide/bisacrylamide (29:1)
- 10% ammonium persulfate (APS)
- Tetramethylethylenediamine (TEMED) 116.24 g mol-1
- Butanol (water-saturated)
- SDS-PAGE running buffer (1X)
- SDS-PAGE sample buffer (4x)
- SDS-PAGE gel casting system
- SDS-PAGE gel running system
- Power supply

Experiment settings
- Number of gels: 1
- Percentage of acrylamide: 7.5 %
- Volume of separation gel: 6 ml

Step 1

Setup the SDS-PAGE gel casting system and check for leakage with water.

Step 2

Prepare 6 ml separation gel ( 7.5 % ) in a disposable tube as follows:

● 1.5 ml separation buffer (4X)


● 1.5 ml 30% acrylamide/bisacrylamiide (29:1)
● 2.9 ml dH2 0

Step 3

Only add directly before casting the gel !!

● 100 µl fresh APS (10%)


● 5 µl TEMED

Step 4

Immediately cast the gel (leave 2-3 cm for the stacking gel) and add 1 ml of butanol (water-saturated)

Step 5

Allow the gel to polymerize for at least 1h

Step 6

Remove the butanol, rinse with some dH2 0 and remove all liquid with some filter paper

Step 7

Prepare the stacking gel for 1 gels by mixing in a disposable tube:


● 0.88 ml stacking buffer (4X)
● 0.56 ml 30% acrylamide/bisacrylamide (29:1)
● 2.01 ml dH2 0

Step 8

Only add directly before casting the gel!!

● 50 µl fresh APS (10%)


● 5 µl TEMED

Step 9

Immediately pipet the stacking gel on top of the polymerized separating gel, insert a comb with the appropriate number of wells and
allow the gel to polymerize for 1h

Step 10

Place the gels in the running system and fill the system with SDS-PAGE running buffer

Step 11

Mix 3 µl of the protein sample with 1 µl of SDS-sample buffer (4X) and boil for 10 min

Step 12

Load the samples on gel and run the gel at 130 V until the blue loading dye reaches the bottom of the gel.

Step 13

Disassemble the running system to take out the gel. Proceed to CBB staining/Silver staining of the gel or Western blotting

Acrylamide is highly neurotoxic!! Prevent any skin contact and always wear gloves!

Based on: Laemmli UK (1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4". Nature

227 (5259): 680-685

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