SDS-PAGE Protein Gels

Consolidated/Pictorial Protocol

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 (and to isolate membranes for NREL analysis)

• Grow 50 ml cells to ~ OD 1.0
• Weigh 50 ml tube and record

- Need to subtract tube weight to determine wet cell weight (WCW) of sample -- don't forget this step!

• Transfer cells from vial to tube and pellet
• Dry the pellet as completely as possible by decanting and then pipetting off any remaining liquid
• Weigh dried pellet and 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

• Resuspend cell pellet (should have 0.2-0.5g wet cell mass) in 3 ml of 50 mM Tris-HCl (pH 7.5) + 0.25M sucrose
• French press at 1000 psi twice
• Centrifuge at 5000 RPM for 10 minutes at 4°C (pellet will be mostly cell debris and mostly brown-pink)
• Transfer supernatant to 50 ml screw-cap Nalgene tube and centrifuge at 14,000 RPM for 20 minutes (pellet will be white)
• Weigh empty ultracentrifuge tubes and record
• Transfer supernatant to ultracentrifuge tubes, balance carefully, and centrifuge at 100,000 x g (= 45K RPM) for 1 hour
• Decant supernatant and dry remaining pellet well
• Weigh pellet and tube, subtract tube weight and record collected membrane weight

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

• If using home-made gels, position so that the shorter plate faces inward!
• Apply pressure on gel holder and gels as you close the tabs to seal the center compartment.

Mini-cell Gel holder

• Fill central compartment with running buffer

Be sure to 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 -80^oC.
• Ladder is stored at -20°C. 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.

Recipes

Destain for Coomassie - 1 L

30% methanol, 10% acetic acid

Directions:

1. Add 100 ml of glacial acetic acid to 600 ml of ddH2O
2. Add 300 ml of methanol and mix
3. Store at room temperature in a sealable container