SDS-PAGE Protein Gels

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(For 5GB1 membrane-separated 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)'''''
==='''''For 5GB1 membrane-separated protein SDS-PAGE'''''===
==='''''For 5GB1 membrane-separated protein SDS-PAGE'''''===

Revision as of 18:49, 20 May 2013


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)

For 5GB1 membrane-separated protein SDS-PAGE

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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