SDS-PAGE Protein Gels

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(For 5GB1 membrane-separated protein SDS-PAGE)
(For 5GB1 membrane-separated protein SDS-PAGE)
Line 24: Line 24:
'''''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)'''''
-
==='''''For 5GB1 membrane-separated protein SDS-PAGE'''''===
+
==='''''For 5GB1 membrane-separated protein SDS-PAGE (and to isolate membranes for NREL analysis)'''''===
-
*Grow cells to ~ OD 1.0
+
*Grow 50 ml cells to ~ OD 1.0
*Weigh 50 ml tube and record
*Weigh 50 ml tube and record
:- Need to subtract tube weight to determine wet cell weight (WCW) of sample -- don't forget this step!
:- Need to subtract tube weight to determine wet cell weight (WCW) of sample -- don't forget this step!
-
*Pellet cells and dry as much as possible
+
*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'''==
=='''Considerations for Loading the Gel'''==

Revision as of 19:17, 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)

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
  • 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.
Personal tools