SDS-PAGE Protein Gels

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(New page: === Consolidated/Pictorial Protocol === SDS casting protocol =='''Sample Prep - Marina Protocol'''== '''''For 5GB1 SDS-PA...)
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Revision as of 17:55, 20 May 2013

Contents

Consolidated/Pictorial Protocol

SDS casting protocol
SDS casting protocol

Sample Prep - Marina Protocol

For 5GB1 SDS-PAGE total protein concentration estimation

  1. 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)
  1. Weigh eppindorf tube
Need to subtract tube weight to determine wet cell weight (WCW) of sample -- don't forget this step!
  1. Harvest appropriate sample volume and pellet cells
RT benchtop centrifugation at 14K RPM; 2-3 minutes should be more than sufficient
  1. Dry the pellet as completely as possible by decanting and then pipetting off any remaining liquid
  2. Weigh dried pellet and eppindorf tube, subtract out tube weight and record WCW


Consolidated/Pictorial Protocol

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

Running the Gel

  • Bio-Rad Mini-Cell Setup
    • If only 1 gel, 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
    • should 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!
  • 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.
    • 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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