Oct 09, 2022

Public workspaceSDS-PAGE

DOI
dx.doi.org/10.17504/protocols.io.kqdg3pm11l25/v1
  • 1Tecnologico de Monterrey Campus Chihuahua
Denisse Chacón Ramírez
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DOI: dx.doi.org/10.17504/protocols.io.kqdg3pm11l25/v1
Protocol Citation: Ana Belem García González, Georgina Diego, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz 2022. SDS-PAGE. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3pm11l25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 08, 2022
Last Modified: October 09, 2022
Protocol Integer ID: 68368
Abstract
This protocol shows the steps carried out by team Tec-Chihuahua to perform SDS-PAGE
Materials
SOLUTIONS:
  • Acrylamide/Bis 30%
  • 29.2 g of Acrylamide.
  • 0.8 g of Bis Acrylamide
  • Dissolve in 50 mL of distilled water in constant agitation, gauge to a volume of 100 mL.
  • Store at 4 °C degrees while protected from light.

  • Tris-HCl/ SDS 4X pH 8.8 (1.5 M Tris-HCl, 0.4% SDS)
  • 18.17 g of TRIZMA® base
  • 0.4 g of SDS
  • Dissolve in 80 mL of distilled water
  • Adjust to a pH de 8.8 with HCl and gauge to a 100 mL
  • Filtrate the solution
  • Store at 4 °C degrees

  • Tris-HCl/ SDS 4X pH 6.8 (0.5 M Tris-HCl, 0.4% SDS)
  • 6.06 g de TRIZMA® base
  • 0.4 g de SDS
  • Dissolve en 80 mL of distilled water.
  • Adjust to a pH de 6.8 with HCl and gauge to a 100 mL
  • Filtrate the solution
  • Store at 4 °C degrees

  • 10% Ammonium Persulfate
  • 500 mg of APS (Ammonium Persulfate)
  • Dissolve en 5 mL of distilled ultra pure water
  • Store at -20 °C for up to two weeks
  • Store at 4 °C degrees for one use only

  • 2X Loading Buffer
  • 2 mL of glycerol
  • 400 uL of Mercapto
  • 0.02 g of bromophenol blue
  • 0.4 g of SDS
  • 2.5 mL of 8.6 pH buffer


  • 1X Running Buffer
  • 14 g of glycine.
  • 3 g of Trizma base.
  • 1 g of SDS.
  • Gauge to a 1 L.


  • Staining Solution
  • 1.25 g of brilliant blue R in 250 mL of methanol
  • 200 mL of distilled water.
  • 50 mL of Glacial Acetic Acid
  • Store in room temperature while protected from light

  • Destaining Solution
  • 250 mL of methanol
  • 62.5 mL of Glacial Acetic Acid
  • 312.5 mL of distilled water.
  • Store in room temperature
Usage:Pour solution into a container and allow to stir until the SDS gel is clear or electrophoresis bands are visible.
Protocol
Protocol
3h 45m
3h 45m
Clean the components of the electrophoresis camera with 70% ethanol and gauze 's.
Assemble the chamber and check that there are no leaks by pouring distilled water between the glasses.
Prepare polyacrylamide gels:


AB
Reagents2 minigels
Distilled water3.4 mL
Acrylamide/Bis 30%4 mL
Tris-HCl/SDS 4X pH 8.8 (1.5 M Tris-HCl, 0.4% SDS)2.5 mL
ASP 10%100 µL
TEMED4 µL
Separation gel (12%)

Pour the solution between the glasses with a 1 mL micropipette, leaving a space of 1.5 cm for the concentrating gel. To level, distilled water is added and allowed to settle for Duration00:30:00 or until a line is seen between the gel and the water.

AB
Reagents2 minigels
Distilled water2.7 mL
Acrylamide/ Bis 30%1 mL
Tris-HCl/ SDS 4X pH 8.8 (1.5 M Tris-HCl, 0.4% SDS)1.3 mL
ASP 10%50 µL
TEMED4 µL
  • Concentrating gel (6%)


30m
Pour solution onto separating gel using a 1 mL micropipette.
Duration00:30:00 Duration00:30:00 Insert the comb (carefully avoiding the formation of bubbles) and leave to solidify for Duration00:30:00

1h 30m
When the gels are polymerized, prepare an electrophoresis chamber with 1X running buffer until it covers the gels and it reaches the line of two gels.
Sample PTake the pellets contained in Eppendorf tubes
  • Take the pellets contained in Eppendorf tubes
  • Add Amount300 µL of 1X loading buffer and resuspend the pellet.reparation:

To denature proteins, heat samples in boiling water for 5 min.
Load gels with the hot sample.
In the first well add Amount7 µL of the molecular weight marker.

Once the samples are loaded, run the gel at 80 V for Duration00:20:00 and then at 180 V for Duration00:45:00

1h 5m
Turn off the camera and disarm it.
Remove the gels from the glasses and place in a container with staining solution. Leave stirring for one hour.
Remove staining solution after one hour, add destaining solution and leave stirring for Duration00:20:00 Change the destaining solution and leave again for Duration00:20:00

40m
Leave stirring until the gel is transparent.
Analyze the gels.