TMIG Standard Methods in Proteomics

The TMIG Standard Procedure of 2-D PAGE in Proteomics
Tosifusa Toda
Proteomics Collaboration Center
Tokyo Metropolitan Institute of Gerontology

Last update : Nov 1, 2007

Please cite the following our papers in your paper if you use our optimized procedures for proteomic analysis.

Jpn. J. Electroph. 1997, 41(1): 13-19. -----------------For oiginal protocol of our 2-D gel electrophoresis
Electrophoresis 1998 Feb;19(2): 344-348 ---------- For 2-D gel protein database
Mech Ageing Dev 1998 Jan 30;100(2): 133-143 --For "on-membrane" protein digestion
J Electrophoresis 2005, 49(4)77-83. --For "in-gel" protein digestion

And we also recommend you to refer to this URL (http://proteome.tmig.or.jp/2D/2DE_method.html), because the procedures shown in this Web site include slight modifications and supplements.

Related papaers on applications of our 2-D gel-based proteome analysis

Electrophoresis 2000 May;21(9): 1814-1822 ------ Application to cell immortalization research
J Electrophoresis 2007, 51(1):1-8.(PDF 1.3 MB) ----- Application to stem cell research

Back to the top page of TMIG-2DPAGE Proteome Database

Apparatus

First dimension : immobilized pH-gradient isoelectric focusing

CoolPhoreStar IPG-IEF (Anatech)

Protean IEF Cell (Bio-Rad)
Second-dimension : SDS-PAGE

CoolPhoreStar SDS-PAGE Tetra-200 (Anatech)

Protean II XL (Bio-Rad)
The source of electric power

PowerPhoreStar Pro3800 (Anatech, Japan)

Powerpack 3000 (Bio-Rad)

Reagents and Consumables

Sigma: T-1503

Urea,

Nakarai Tesque: Code No. 35940-81

Thiourea,

Sigma: T-7875

CHAPS,

Sigma: C-5849

Tricine,

Sigma: T-0377

Sulfobetain 10 (SB3-10),

Amresco: J548-10G

Orange G,

Sigma: O-1625

Electrofocusing sample application pieces,

Amersham Pharmacia Biotech: Code No. 80-1129-46

or Anatech: Code No. 3600-13

Electrofocusing electrode wicks,

Amersham Pharmacia Biotech: Code No. 18-1004-40

or Anatech: Code No. 3600-12)

Silver stainning kit,

Wako Pure Chem.: Code No. 299-13841

Silcon oil 5c/s,

Shin-Etsu Chem.: Code No. KF-96-L-5CS

pI Marker protein kit,

Daiichi Chem.: Code No. 220760

Molecular weight marker protein kit,

Daiichi Chem.: Code No. 181122 or 181066

Gel strip for the first-dimensional IPG-IEF (Immobilized pH gradient isoelectric focusing):
Rehydrate Immobiline DryStrip (Amersham Pharmacia Biotech) or IPG ReadyStrip (Bio-Rad) using a rehydration solution, such as shown below:

urea                  14.4 g
thiourea               6.1 g
dithiothreitol         0.08 g
Pharmalyte 3-10        0.4 ml
0.1 M acetic acid      1.0 ml
0.1%(w/v) Orange G     1.0 ml
20%(v/v) Triton X-100  4.0 ml
pure water     up to  40.0 ml

The volume is for 8 strips of 18-cm or 17-cm IPG gel (5 ml per strip).

Gel plates for the second-dimensional SDS-PAGE:

35 %(w/v) acrylamide         106  ml
 2 %(w/v) BIS-acrylamide      58  ml
1.5 M     Tris-HCl, pH 8.8   124  ml
60%(v/v)  glycerol            90  ml
pure water    up to          500  ml
--- degas in vacuo before mixing reagents shown below---
10 %(w/v) SDS                 5.0 ml
10 %(w/v) ammonium persulfate 1.0 ml
TEMED                         0.3 ml

The volume is for 8 plates of 7.5%T, 3%C polyacrylamide gel (190 x 180 x 1 mm).

Preparation of protein extraction reagents:


   For cytosolic/hydrophilic proteins       For membrane/particulate-bound proteins
   Urea                   0.51  g           Urea                   0.30  g
   10%(w/v) SDS           0.02 ml           thiourea               0.15  g
   10%(v/v) Triton X-100  0.20 ml           20%(w/v) CHAPS         0.10 ml
   DTT                    0.01  g           20%(w/v) SB3-10        0.10 ml
   Pharmalyte 3-10        0.02 ml           DTT                    0.01  g
   Milli-Q water    up to 1.00 ml           Pharmalyte 3-10        0.02 ml
                                            Milli-Q water    up to 1.00 ml

   [Supplements for preventing protein degradations completely (Optional)]
    Sigma Proteinase inhibitor Cocktail   10 ul/ml
    Sigma Phosphatase Inhibitor I          5 ul/ml
    Sigma Phosphatase Inhibitor II         5 ul/ml

Preocedure of protein extraction from tissue or packed cell pellet:

Suspend tissue fragments or cells on 3 volumes of Protein Extraction Reagent A or B.
Disrupt cells by ultrasonication for 1 sec x 20 times.
Remove the supernatant by centrifugation at 15,000 rpm for 20 min at room temperature.
Store small aliquots of the supernatant below -70C until use for electrophoresis. (Incubate for 3 min at 45C prior to application)

Protein extraction from cell cultures (Alternative ):

Rinse the culture dish with 5 ml of PBS- for 1 min, repeat 3 times.
Scrape off cells from the culture dish on ice by rubbing with a plastic scraper.
Transfer the cell suspension to a 1.5-ml microfuge tube of which tare was weighed in advance. (Give W [mg]= the weight of the cell suspension)
Add W x 0.85 mg of urea, W x 0.05 micro-liter 2-mercaptoethanol, W x 0.1 micro-liter of 1.5%(w/v) SDS, 8.5%(v/v) Triton X-100.
Disrupt cells by ultrasonication for 1 sec x 20 times.
Remove the supernatant by centrifugation at 15,000 rpm for 20 min at room temperature.
Store small aliquots of the supernatant below -70C until use for electrophoresis. (Incubate for 3 min at 45C prior to application)

Procedure of the first-dimensional IPG-IEF (Immobilized pH-gradient isoelectric focusing):

Pour a small volume of silicon oil into the electrophoresis chamber, mount Immobiline DryStrip supporting plate in the chamber giving care to remove air bubbles beneath the plate.
Add 1 micro-liter of pI marker protein solution to an aliquot (10-20 micro-liter) of cell extract if neccessary. (In the case of "DAIICHI" 2D pI Marker for silver staining, the content of a vial is dissolved in 0.5 ml of pure water. The reconstituted solution is storable at -20C ).
Stand re-swelled Immobiline Dry Strip on its long edge on filterpaper briefly to remove excess Gel Swelling Solution, then lay on the filterpaper gel-side-up.
Put a piece of Sample Application Filter on the gel ca. 1-cm inside from the cathodic* end. Apply the cell extract (10-20 micro-liter) to the Sample Application Filter.
(* This is for pH 4-7 and 3-10 Drystrips. Anodic application is better for pH 6-11 Drystrip)
Mount the strip in the electrophoresis chamber which is pre-cooled to 20C.
Put electrode pads wetted with distilled water on both ends of the strip to recieve sure contact of platinum electrodes.
Set the platimum electrode, and cover the gel strip and electrode pads with silicon oil (5 c/s) to isolate electrophoresis media from the atmosphere.

Apply electric power in the stepwise elevation of volutage as follows:



For pH 4-7 and 3-10 Drystrips		For pH 6-11 DryStrip  For pH 5.0-6.0 DryStrip

	(18-cm long)			(18-cm long)			(18-cm long)

 Step	Voltage	Time (h)		Voltage	Time (h)		Voltage	Time (h)

  1	 500 V	 2 h			 350 V	8 h			 500 V	 1.0 h
  2	 700 V	 1 h			 500 V	1 h			 700 V	 0.5 h
  3	1000 V	 1 h			1000 V	1 h			1000 V	 0.5 h
  4	1500 V	 1 h			1500 V	1 h			1500 V	 0.5 h
  5	2000 V	 1 h			2000 V	1 h			2000 V	 0.5 h
  6	2500 V	 1 h			2500 V	1 h			2500 V	 0.5 h
  7	3000 V	 1 h			3000 V	1 h			3000 V	 0.5 h
  8	3500 V	10 h			3500 V	3 h			3500 V	13.0 h

Keep the electric power supply at 500 V after the step 8.

Treatment of the gel strip for the second-dimensional SDS-PAGE:

1. Primary treatment for disulfide reduction.

Incubate the gel strip for 30 min at room temperature in the SDS-treatment solution prepared as follows.


SDS-treatment solution for 8 strips:

urea                      14.5  g
dithiothreitol             0.2  g
0.5 M  Tris-HCl, pH 6.8    2.0 ml
10%(w/v) SDS stock         8.0 ml
0.1%(w/v) BPB	           1.0 ml   
60%(v/v) glycerol         20.0 ml

2. Secondary treatment for alkylation.

Incubate the gel strip for 20 min at room temperature in one of the following alkylation reagents.


Carbamoylmethylation (CM) Reagent for 8 strips:

0.5 M  Tris-HCl, pH 6.8    2.0 ml
10%(w/v) SDS stock         8.0 ml
0.1%(w/v) BPB              1.0 ml
60%(v/v) glycerol         20.0 ml
Milli-Q water              9.0 ml
iodoacetamide              1.8 g             ICH₂CONH₂
                       --> Protein-S-CH₂-CO-NH₂



Pyridylethylation (PE) Reagent for 8 strips: 

0.5 M  Tris-HCl, pH 6.8    2.0 ml
10%(w/v) SDS stock         8.0 ml
0.1%(w/v) BPB              1.0 ml
60%(v/v) glycerol         20.0 ml
Milli-Q water              9.0 ml
4-vinylpyridine            1.0 ml         CH₂=CH-Pyridyl

                       --> Protein-S-CH₂-CH₂-Pyridyl

Procedure of the Second-dimensional SDS-PAGE

Pour an appropriate volume of the anodic electrode solution into the vertical electrophoresis chamber.
Incubate the gel strip in SDS-treatment solution for 5 min at room temperature again.
Fill the top of the gel slab with the cathode solution, place the gel strip that is trimmed to fit the gel size, and gently push the strip down using a shark tooth-shaped Strip Fixer Comb to achieve the firm contact to the gel slab.
Mount the gel plate on the electrophoresis chamber unit.
Fill the cathodic chamber with the cathodic electrode buffer.
Perform electresis in 20-40 mA/slab until the BPB dye comes near the gel bottom.
Proceed to the subsequent protein staining or an electro-transfer blotting.

Electrode Buffer Solutions for SDS-PAGE:
Preparation of 10x stock of anodic electrode buffer (2 M Tris-HCl, pH 8.8):


Tris      242.2 g
in 700 ml of Milli-Q SP water.

Adjust pH to 8.8 with 6N HCl
Add pure water upto 1,000 ml

Preparation of 5x stock of cathodic electrode buffer (Tris-Tricine Buffer ):


Tris      60.5 g
Tricine   89.5 g
SDS        5.0 g

in 1,000 ml of pure water.

Pro-Q Diamond Staining for phosphoprotein detection

Immerse the gel in 50%(v/v) methanol, 10%(v/v) acetic acid and incubate for at least 30 min at room temperature.
Repeat the fixaton step once more to remove SDS from the gel completely. Gel can be left in the fixing solution overnight.
Rinse the gel in Milli-Q water for 10 min.
Repeat the rinsing once more to remove methanol and acetic acid completely. Residual methanol or acetic acid will interfere with Pro-Q Diamind staining.
Immerse the gel in Pro-Q Diamond phosphoprotein gel stain with gentle agitation for 75-120 min in the dark.
Rinse the gel in the Destain Solution for 1 h in the dark.
Repeat the rinsing once more.

For preparation of Destain Solution:
- Add 50 ml of 1 M sodium acetate, pH 4.0, and 200 ml of acetonitrile to 750 ml of Milli-Q water.

SYPRO Ruby Staining

Incubate the gel in 50%(v/v) methanol, 10%(v/v) acetic acid for more than 30 min. Gel can be left in the fixing solution overnight.
Incubate the gel in 10%(v/v) methanol, 7%(v/v) acetic acid for 30 min.
Immerse the gel in the dark in SYPRO Rubu gel stain with gentle agitation for 90-120 min.
Rise the gel in 10%(v/v) methanol, 7%(v/v) acetic acid for 30 min.

Coomassiee Blue Staining (optinal)

Incubate the gel in 50%(v/v) methanol, 10%(v/v) acetic acid for more than 30 min.
Incubate the gel in working reagent of Quick-CBB Protein Stain Reagent Kit (299-50101, Wako Pure Chemicals) for 30 min - 1 h.
Rise the gel in 7%(v/v) acetic acid.

Silver staining (optinal)

In-Gel Protein Digestion for Peptide Mass Fingerprinting

Pick a small gel piece up from a protein spot, and put it in a 96-well PCR plate (ABGene Thermo-Fast 96, Skirted).
Immerse the gel piece in 0.1 ml of Reducing Reagent for 30 min.

Reducing Reagent :1.5 mg/ml dithiothreitol in 100 mM ammonium bicarbonate
Immerse the gel piece in 0.1 ml of Alkylating Reagent for 30 min.
Alkylating Reagent :10 mg/ml iodoacetamide in 100 mM ammonium bicarbonate
Immerse the gel piece in 0.1 ml of 50% methanol, 50mM ammonium bicarbonate for 15 min x 2.
Immerse the gel piece in 0.1 ml of 50% acetonitrile, 50mM ammonium bicarbonate for 10 min x 3.
Immerse the gel piece in 0.1 ml of 100% acetonitrile for 5 min.
Remove the liquid completely and dry up the gel.
Reswell the gel piece in 20 ul of the Digestion Reagent.

Digestion Reagent: 5 ug/ml sequencing grade modified trypsin (Promega V511A or Sigma T-6567),

in 30%(v/v) acetonitrile, 50 mM ammonium bicarbonate.
Seal up the PCR plate with an adhesive seal (SUMILON Plate Seal MS-30010, Sumitomo Bakelight Co. Ltd.) to prevent evaporation.
Incubate the gel piece in the digestion reagent overnight at 30C.

* Extra extraction procedure is not necessarily required for MALDI-TOF/MS.
Sufficient extraction of digested peptides into the digestion reagent has been finished during the overnight incubation in our digestion reagent. The digestion reagent after incubation is applicable to a MALDI target plate by direct mixing with an equivalent volume of matrix solution.
* Step 2 and 3 can be skipped if the sample protein has already been alkylated before 2-DE.

Re-solubilization of Stained Proteins in the Gel and Electrotransfer blotting to PVDF membrane (An alternative procedure to the in-gel digestion)*

Rinse the gel in pure water, for 15 min x 3 times.
Incubate in the re-solubilization reagent (0.3%(w/v) Tris, 0.7%(w/v) glycine, 0.2%(w/v) SDS) for 15 min.
Overlay with Sequi-Blot PVDF membrane (Bio-Rad), and mount in a blotting chamber with the electrotransfer buffer (0.3%(w/v) Tris, 1.5%(w/v) glycine, 0.1%(w/v) SDS)
Apply 4 V/cm in the electrotransfer buffer (0.3%(w/v) Tris, 1.5%(w/v) glycine, 0.1%(w/v) SDS) overnight.

*Please cite our previous paper "Proteomic analysis of Epstein-Barr virus-transformed human B-lymphoblastoid cell lines before and after immortalization" Toda T, Sugimoto M, Omori A, Matsuzaki T, Furuichi Y, Kimura N., Electrophoresis. 2000 May;21(9):1814-22, if you use the procedure for electrotransfer blotting after staining of protein in your research.

On-Membrane Protein Digestion

Punch out a small area of protein spot from a stained Sequi-Blot PVDF membrane (Bio-Rad).
Put it in a microfuge tube siliconized with SIGMACOTE^TM.
Destain the membrane with 60%(v/v) methanol, 10%(v/v) acetic acid (15 min x 3).
Bufferize the membrane in 2.5 mM Tris-HCl, pH 7.7 (3 min x 3).
Treat the membrane with 4.5%(w/v) Polyvynyl pirolidone 25, 2.5 mM Tris-HCl, pH 7.7 for 30 min.
Wash the membrane with pure water (3 min x 3).
Wash the membrane with 5%(v/v) acetonitrile (1 min x 3).
Dip the membrane in 30 micro-l of 8%(v/v) acetonitrile, 2.5 mM Tris-HCl, pH 7.7.
Incubate the membrane in 20 micro-l of 0.15 U/ml Endoproteinase Lys-C (Boehringer Cat#13832451) diluted in 8%(v/v) acetonitrile, 2.5 mM Tris-HCl, pH 7.7 overnight at 30C.
Add 20 micro-l of acetonitrile, and incubate for more 1 h at room temperature.
Remove the peptide solution to another tube, and desalt using a ZipTip for MS analysis or microsequencing (optional).

Desalting with ZipTip (optional)

Evoporate the solvent using SpeedVac or under a flow of nitrogen gas.
Dissolve peptides in 10 micro-l of 0.1% TFA.
Adsorb peptides on ZipTip C18 resin (Millipore), that has been previously prewetted with 50% acetonitrile and equilibrated with 0.1% TFA.
Wash the resin with 10 micro-l of 0.1% TFA by 5 strokes of pumping.
Elute peptides into 5 micro-l of 50% acetonitrile, 0.1% TFA.
Mix 1 micro-l of peptide solution with 1 micro-l of matrix solution, and apply to a target of MALDI-TOF/MS.

Matrix solution for MALDI-TOF/MS


Alpha-cyano-4-hydroxycinnamic acid (Sigma C-2020)   10  mg
Acetonitrile (HPLC grade)                            0.5 ml
Methanol    (HPLC grade)                            0.4 ml
TFA (Sigma T-6508)                                  1 µl
Milli-Q water                                       0.1 ml

All rights are reserved by T. Toda (2000-2007)
Most part of this protocol is based on our standardized procedure published in Jpn. J. Electroph. 1997, 41(1), 13-19. However, the current version has been slightly modified for optimization in proteomics after publication. Please cite this URL (http://proteome.tmig.or.jp/2D/2DE_method.html) when you refer to our prodedure in your publication.