SDS-PAGE Protein Gels

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=== Consolidated/Pictorial Protocol ===
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[[image:SDS-PAGE sample prep and loading.jpg|thumb|upright=3.0|center|SDS-PAGE sample prep and loading]]
=='''Sample Prep - Marina Protocol'''==
=='''Sample Prep - Marina Protocol'''==
'''''For 5GB1 whole cell protein SDS-PAGE'''''
'''''For 5GB1 whole cell protein SDS-PAGE'''''
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*Incubate sample for 5 minutes at 95-100°C
*Incubate sample for 5 minutes at 95-100°C
'''''Samples are ready to be loaded into gel as described below'''''
'''''Samples are ready to be loaded into gel as described below'''''
 +
'''''As of May 2013 5GB1 proteins are analyzed using Bio-Rad Mini-Protean TGX Precast Gels, 12% polyacrylamide (Cat# 456-1045)'''''
'''''As of May 2013 5GB1 proteins are analyzed using Bio-Rad Mini-Protean TGX Precast Gels, 12% polyacrylamide (Cat# 456-1045)'''''
-
=== Consolidated/Pictorial Protocol ===
+
=='''Considerations for Loading the Gel'''==
-
[[image:SDS-PAGE sample prep and loading.jpg|thumb|upright=3.0|center|SDS-PAGE sample prep and loading]]
+
* Note that gels are both concentration- and volume-dependent; overloading of either will cause deformation of lanes and smearing
 +
* 15 ul of 5GB1 prepared as above gives pretty good banding and a concentration ~4ug/ul
 +
* Use 5 ul ladder
 +
* Use BSA (usually comes at [10 ug/ul]) to make standards for calibration curve
=='''Running the Gel '''==
=='''Running the Gel '''==
*Bio-Rad Mini-Cell Setup
*Bio-Rad Mini-Cell Setup
-
**If only 1 gel, use buffer dam to replace second gel
+
*Use diluted 10X Tris/Glycine/SDS Buffer
 +
::Re-using running buffer 2-3 times does not affect results
 +
::If only running one gel, don't forget to use buffer dam to replace second gel
*'''Position the gels with the shorter plate facing inward!'''
*'''Position the gels with the shorter plate facing inward!'''
* Apply pressure on gel holder and gels as you close the tabs to seal the center compartment.
* Apply pressure on gel holder and gels as you close the tabs to seal the center compartment.
[[image:gel holder.jpg|thumb|center|Mini-cell Gel holder]]
[[image:gel holder.jpg|thumb|center|Mini-cell Gel holder]]
* Fill central compartment with running buffer
* Fill central compartment with running buffer
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** should fill sample wells
+
:: Pour enough to fill sample wells!
* Pour more into the outer compartment to specified line
* Pour more into the outer compartment to specified line
* Load gel
* Load gel
-
** Make sure you will be able to determine the orientation of your gel after it is stained.  Asymmetry is good!
+
:: Make sure you will be able to determine the orientation of your gel after it is stained.  Asymmetry is good!
-
* Make sure to color/charge-match the cords to the power unit as the electrodes in the gel holder to the contacts in the lid.
+
* Run @ 60 V for ~15 min, then 200 V for ~ 20+ min.
* Run @ 60 V for ~15 min, then 200 V for ~ 20+ min.
-
** Note: ladder looks blurry while running through the stacking gel; don't be alarmed unless it still looks blurry in the resolving gel.
+
:: Note: ladder looks blurry while running through the stacking gel; don't be alarmed unless it still looks blurry in the resolving gel.
-
**Can skip the 60V step if you don't need a gorgeous gel
+
::Can skip the 60V step if you don't need a gorgeous gel
-
** Amanda runs 20 min at 200V, then checks frequently to make sure the protein doesn't run off the gel.
+
::Amanda runs 20 min at 200V, then checks frequently to make sure the protein doesn't run off the gel.
==Storing Samples After Use==
==Storing Samples After Use==
* Amanda was taught to flash-freeze (liquid N<sub>2</sub>) samples and store them at -80<sup>o</sup>C.  Janet loves liquid N2 (particularly dumping it on the ground after use) so I follow along without question.
* Amanda was taught to flash-freeze (liquid N<sub>2</sub>) samples and store them at -80<sup>o</sup>C.  Janet loves liquid N2 (particularly dumping it on the ground after use) so I follow along without question.
* Ladder is stored at -80oC.  An aliquot of PageRuler Prestained that sat out at room temp for 1 week looked perfect in a gel, so don't worry about that ladder's stability.
* Ladder is stored at -80oC.  An aliquot of PageRuler Prestained that sat out at room temp for 1 week looked perfect in a gel, so don't worry about that ladder's stability.

Revision as of 18:39, 20 May 2013

Contents

Consolidated/Pictorial Protocol

SDS-PAGE sample prep and loading
SDS-PAGE sample prep and loading

Sample Prep - Marina Protocol

For 5GB1 whole cell protein SDS-PAGE

  • Grow cells to ~ OD 1.0
- For calculation purposes, you ideally want 1 ml of cells at an OD of 1.0
- If your samples are not at 1.0, increase or decrease volume to compensate (eg: If OD is only 0.747, use 1.34 ml)
  • Weigh eppindorf tube
- Need to subtract tube weight to determine wet cell weight (WCW) of sample -- don't forget this step!
  • Harvest appropriate sample volume and pellet cells
- RT benchtop centrifugation at 14K RPM; 2-3 minutes should be more than sufficient
  • Dry the pellet as completely as possible by decanting and then pipetting off any remaining liquid
  • Weigh dried pellet and eppindorf tube, subtract out tube weight and record WCW

At this point, pellet can be frozen and the following steps can be performed at a later date

  • Mix Laemmli sample buffer using the following recipe:
500 ul Bio-Rad 4x Laemmli buffer
450 ul dH2O
50 ul 2-mercaptoethanol
  • Add 100 ul of buffer to pellet and vortex to ensure cells and Laemmli buffer are well mixed
  • Incubate sample for 5 minutes at 95-100°C

Samples are ready to be loaded into gel as described below

As of May 2013 5GB1 proteins are analyzed using Bio-Rad Mini-Protean TGX Precast Gels, 12% polyacrylamide (Cat# 456-1045)


Considerations for Loading the Gel

  • Note that gels are both concentration- and volume-dependent; overloading of either will cause deformation of lanes and smearing
  • 15 ul of 5GB1 prepared as above gives pretty good banding and a concentration ~4ug/ul
  • Use 5 ul ladder
  • Use BSA (usually comes at [10 ug/ul]) to make standards for calibration curve

Running the Gel

  • Bio-Rad Mini-Cell Setup
  • Use diluted 10X Tris/Glycine/SDS Buffer
Re-using running buffer 2-3 times does not affect results
If only running one gel, don't forget to use buffer dam to replace second gel
  • Position the gels with the shorter plate facing inward!
  • Apply pressure on gel holder and gels as you close the tabs to seal the center compartment.
Mini-cell Gel holder
Mini-cell Gel holder
  • Fill central compartment with running buffer
Pour enough to fill sample wells!
  • Pour more into the outer compartment to specified line
  • Load gel
Make sure you will be able to determine the orientation of your gel after it is stained. Asymmetry is good!
  • Run @ 60 V for ~15 min, then 200 V for ~ 20+ min.
Note: ladder looks blurry while running through the stacking gel; don't be alarmed unless it still looks blurry in the resolving gel.
Can skip the 60V step if you don't need a gorgeous gel
Amanda runs 20 min at 200V, then checks frequently to make sure the protein doesn't run off the gel.

Storing Samples After Use

  • Amanda was taught to flash-freeze (liquid N2) samples and store them at -80oC. Janet loves liquid N2 (particularly dumping it on the ground after use) so I follow along without question.
  • Ladder is stored at -80oC. An aliquot of PageRuler Prestained that sat out at room temp for 1 week looked perfect in a gel, so don't worry about that ladder's stability.
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