Buffer Formulations - Bio-Rad Laboratories

Life Science Group Bulletin 6199 Rev A US/EG Bio-Rad Laboratories, Inc. 11-0864 1111 Sig 1211 Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699

Life Science Group Bulletin 6199 Rev A US/EG Bio-Rad Laboratories, Inc. 11-0864 1111 Sig 1211 Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00

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Buffer Formulations

Protocol
Bulletin 6199

Sample Preparation Buffers
1 M Tris-HCl, pH 7.6 (100 ml)
Tris base Deionized H2O (diH2O) Adjust pH to 7.6 with HCl diH2O
0.5 M Tris-HCl, pH 6.8 (100 ml) (catalog #161-0799)
Tris base diH2O Adjust to pH 6.8 with HCl diH2O Store at 4°C

12.11 g 80 ml to 100 ml
6.06 g ~60 ml to 100 ml

10% SDS (10 ml) (catalog #161-0416)

SDS diH2O

1.00 g to 10 ml

1.0% Bromophenol Blue (10 ml) (catalog #161-0404)
Bromophenol blue diH2O

100 mg to 10 ml

RIPA Solubilization Buffer (100 ml) 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% NP-40 or 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS

NaCl EDTA NP-40 or Triton X-100 Sodium deoxycholate SDS diH2O 1 M Tris-HCl, pH 7.6 diH2O

0.88 g 0.15 g 1 g 1 g 0.10 g 80 ml 2.5 ml to 100 ml

Phosphate Buffered Saline (PBS, 1 L) 0.9% (w/v) sodium chloride in 10 mM phosphate buffer, pH 7.4

NaCl KCl Na2HPO4 KH2PO4 diH2O Adjust pH to 7.4 with HCl or NaOH diH2O

8.00 g 0.20 g 1.44 g 0.24 g 800 ml
to 1 L

SDS-PAGE Sample Buffer (2×, 8 ml) (catalog #161-0737, 30 ml)
62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue

0.5 mM Tris-HCl, pH 6.8

1.0 ml

25% Glycerol

2.0 ml

1.0% Bromophenol blue

0.08 ml

10% SDS

1.6 ml

diH2O

2.92 ml

Store as 1­2 ml aliquots at ­70°C and add b-mercaptoethanol (0.4 ml) or 3% DTT immediately before use.

Protein Precipitation Solution (100 ml) 20% Trichloroacetic acid (TCA), 0.2% DTT in ice-cold acetone (­20°C)

TCA DTT Acetone Dissolve Acetone
Store at ­20°C

20.00 g 0.20 g 80 ml
to 100 ml

Wash Solution (100 ml) 0.2% DTT in ice-cold acetone (­20°C)
DTT Acetone Dissolve Acetone
Store at ­20°C

0.20 g 80 ml
to 100 ml

Lysis Buffer (50 ml) 2 M thiourea, 7 M urea, 4% (w/v) CHAPS, 1% (w/v) DTT, 2% (v/v) carrier ampholytes (pH 3­10)

Urea Thiourea diH2O CHAPS Bio-Lyte® ampholytes DTT diH2O

21.00 g 7.60 g to 45 ml 2.00 g 1.0 ml 0.50 g to 50 ml

SDS Sample Solubilization Buffer (50 ml) 1% (w/v) SDS, 100 mM Tris-HCl (pH 9.5)

SDS Tris base diH2O Titrate to pH 9.5 with diluted HCl diH2O

0.50 g 0.60 g 40 ml
to 50 ml

Gel Casting Reagents

Acrylamide/Bis (30% T, 2.67% C)

Acrylamide (29.2 g/100 ml) N'N'-bis-methylene-acrylamide diH2O

87.60 g 2.40 g to 300 ml

Filter and store at 4ºC in the dark (30 days).

Premade alternatives: Catalog #161-0125, 37.5:1 acrylamide/bis powder Catalog #161-0158, 30% acrylamide/bis solution

1.5 M Tris-HCl, pH 8.8 (150 ml) (catalog #161-0798, 1 L)
Tris base (18.15 g/100 ml) diH2O Adjust to pH 8.8 with 6 N HCl diH2O
Store at 4ºC

27.23 g 80 ml
to 150 ml

0.5 M Tris-HCl, pH 6.8 (catalog #161-0799, 1 L)
Tris base diH2O Adjust to pH 6.87 with 6 N HCl diH2O
Store at 4ºC

6.00 g 60 ml
to 100 ml

10% (w/v) SDS (100 ml) (catalog #161-0416)

SDS diH2O Dissolve with gentle stirring diH2O

10.00 g 90 ml
to 100 ml

10% (w/v) APS (fresh daily) (catalog #161-0416)
Ammonium persulfate diH2O

0.10 g 1 ml

Water-Saturated n-Butanol

n-Butanol

50 ml

diH2O

5 ml

Combine in a bottle and shake. Use the top phase only. Store at

room temperature.

Sample Buffers

2× SDS-PAGE (Laemmli, 30 ml) (catalog #161-0737)
62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 5% b-mercaptoethanol (added fresh)

0.5 M Tris-HCl, pH 6.8

3.75 ml

50% Glycerol

15.0 ml

1.0% Bromophenol blue

0.3 ml

10% SDS

6.0 ml

diH2O

to 30 ml

b-mercaptoethanol (50 l to 950 l sample buffer) before use.

2× Native PAGE (30 ml) (catalog #161-0738)
62.5 mM Tris-HCl, pH 6.8, 40% glycerol, 0.01% bromophenol blue

0.5 M Tris-HCl, pH 6.8 50% Glycerol 1.0% Bromophenol blue diH2O

3.75 ml 24.0 ml 0.3 ml to 30 ml

2× Tricine (30 ml) (catalog #161-0739)
200 mM Tris-HCl, pH 6.8, 2% SDS, 40% glycerol, 0.04% Coomassie Brilliant Blue G-250, 2% b-mercaptoethanol (added fresh)

0.5 M Tris-HCl, pH 6.8

12.0 ml

50% Glycerol

24.0 ml

10% SDS

6.0 ml

Coomassie Blue G-250

12.00 mg

diH2O

to 30 ml

b-mercaptoethanol (20 l to 980 l sample buffer) before use.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6199

Running Buffers

10× SDS-PAGE (1 L) (cata log #161-0732)

250 mM Tris, 1.92 M glycine, 1% SDS, pH 8.3

Tris base

30.30 g

Glycine

144.10 g

SDS

10.00 g

diH2O

to 1 L

Do not adjust the pH (~pH 8.3).

10× Native PAGE (1 L) (catalog #161-0734)
250 mM Tris, 1.92 M glycine, pH 8.3 Tris base Glycine diH2O Do not adjust the pH (~pH 8.3).

30.30 g 144.10 g to 1 L

10× Tris-Tricine (1 L) (catalog #161-0744)
1 M Tris, 1 M Tricine, 1% SDS, pH 8.3 Tris base Tricine SDS diH2O
Do not adjust the pH (~pH 8.3).

121.10 g 179.20 g 10.00 g to 1 L

Buffer Components
0.5 M Tris-HCl, pH 6.8 (1 L) (catalog #161-0799)
Tris base diH2O Adjust to pH 6.8 with HCl diH2O Store at 4°C
10% SDS (250 ml) (catalog #161-0416)
SDS diH2O
1.0% Bromophenol Blue (10 ml) (catalog #161-0404)
Bromophenol blue diH2O

60.60 g ~900 ml to 1 L
25.00 g to 250 ml
100 mg to 10 ml

This is an excerpt from Bio-Rad's comprehensive Electrophoresis Guide (Bulletin 6640).

Bio-Rad Laboratories, Inc.

Life Science Group

Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 9532 220 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 Thailand 800 88 22 88 United Kingdom 020 8328 2000

Bulletin 6199 Rev A US/EG

11-0864 1111 Sig 1211