试剂配制

Tris-buffered sucrose solution

Reagent Final concentration Quantity Tris-base (FW 121.1) 10mM 0.605g Sucrose (FW 342.3) 250mM 42.79g Milli-Q water — 450ml HCl (FW 36.46) adjust to pH7.0 Milli-Q water Make up to 500ml Store at 4℃.

Standard lysis buffer

Reagent Final concentration Quantity Urea (FW 60.06) 7M 10.51g Thiourea (FW 76.12) 2M 3.8g

Tris-base 30mM 0.09g CHAPS (FW 614.89) 4%(w/v) 1g Milli-Q water — 20ml HCl adjust to pH8.5 Milli-Q water Make up to 25ml Small aliquots can be stored at －80℃, for up to 3 months.

Lysine solution (10mM)

Reagent Final concentration Quantity L-Lysine (FW 182.65) 10mM 0.0183g Milli-Q water Make up to 10ml Store in 1ml aliquots at －20℃.

Rehydration stock buffer

Reagent Final concentration Quantity Urea 7M 10.51g Thiourea 2M 3.8g CHAPS 4%(w/v) 1g Milli-Q water Make up to 25ml Small aliquots can be stored at －80℃, for up to 6 months.

Bromophenol blue solution (1%)

Reagent Final concentration Quantity Bromophenol blue 1% 100mg

Tris-base 50mM 60mg Milli-Q water Make up to 10ml Store at room temperature.

Tris-HCl solution (1.5M, pH8.8)

Reagent Final concentration Quantity Tris-base 1.5M 181.7g Milli-Q water 750ml

HCl adjust to pH8.8 Milli-Q water Make up to 1000ml Filter solution through a 0.45μm filter. Store at 4℃. SDS equilibration buffer

Reagent Final concentration Quantity Tris-HCl(1.5M, pH8.8) 50mM 13.4ml

Urea 6M 144.14g Glycerol (87% v/v) 30% (v/v) 138ml SDS (FW 288.38) 2% (w/v) 8g Bromophenol blue (1%) 0.002%(w/v) 800μl Milli-Q water Make up to 400ml Store in 50ml aliquots at －20℃.

Bind-Silane solution（全部用进口试剂）

Reagent Final concentration Quantity Ethanol 80% (v/v) 40ml Glacial acetic acid 2% (v/v) 1ml

Bind-Silane 0.1% (v/v) 50μl Milli-Q water — 9ml Store at room temperature. SDS solution (10%)

Reagent Final concentration Quantity SDS 10% (w/v) 5.0g Milli-Q water Make up to 50ml Filter solution through a 0.45μm filter. Store at room temperature.

Ammonium persulfate, APS (10%)

Reagent Final concentration Quantity 10% (w/v) 0.1g APS (FW 228.20)

Milli-Q water Make up to 1ml Prepare fresh on day of use then discard.

Monomer stock solution (30%T, 2.6% C)

(30% acrylamide, 0.8% N,N’-methylenebisacrylamide)

Reagent Acrylamide (FW 71.08)

N,N’-methylenebisacrylamide (FW154.17) Milli-Q water

Final concentration

30% (w/v)

0.8% (w/v)

Make up to 1000ml

Quantity 300g 8g

Filter solution through a 0.45μm filter. Store at 4℃ in the dark.

SDS electrophoresis buffer (10×)

Reagent Final concentration Quantity Tris-base 25mM 30.3g Glycine (FW 75.07) 192mM 144.0g

SDS 0.1% (w/v) 10.0g Milli-Q water Make up to 1000ml Filter solution through a 0.45μm filter. Store at room temperature.

Gel storage buffer

Reagent Final concentration Quantity Tris-HCl (1.5M, pH8.8) 375mM 50ml

SDS 0.1% (w/v) 0.2g Milli-Q water Make up to 200ml Store at 4ºC.

Water saturated butanol

Reagent Final concentration Quantity Butan-2-ol — 50ml Pure water — 50ml or more Store at room temperature.

Shake to mix and once completely separated, use the top layer to overlay gels.

Agarose sealant

Reagent

Agarose (low melting)

Cathodal electrophoresis buffer Bromophenol blue (1%)

Final concentration Quantity

0.5% (w/v) 0.125g

— 25ml 0.002% (w/v) 50μl

Prepare just prior to use. Add the agarose and bromophenol blue solution into the fresh cathodal electrophoresis buffer (2× or 3×), and heat in boiling water until the agarose is completely dissolved.

Upper chamber agarose sealant

Reagent

Agarose (low melting)

Cathodal electrophoresis buffer Bromophenol blue (1%)

SDS-PAGE resolving gel

Gel Concentration 5% 7.5% 10% 12.5% 15% Monomer stock solution 16.7ml25ml 33.3ml41.7ml 50ml Tris-HCl (1.5M, pH8.8) 25ml 25ml 25ml 25ml 25ml SDS (10%) 1ml 1ml 1ml 1 ml 1ml Milli-Q water 56.8ml48.5ml40.2ml31.8ml 23.5mlAmmonium persulfate (10%) 500μl 500μl 500μl 500μl 500μl TEMED 33μl 33μl 33μl 33μl 33μl Total volume 100ml 100ml 100ml 100ml 100ml 12.5% SDS-PAGE gel composition for Ettan DALT six.

Reagent

Monomer stock solution Tris-HCl (1.5M, pH8.8) SDS (10%) Milli-Q water

Ammonium persulfate (10%) TEMED Total volume

Final concentration Quantity

1% (w/v) 0.5g

— 50ml 0.002% (w/v) 100μl

Volume of reagents for different number of gels

1 2 3 4 5 6 50.04ml75.06ml100.08ml125.1ml150.12ml 175.14ml30ml 45ml 60ml 75ml 90ml 105ml1.2ml0.6ml39.6µl120ml

1.8ml0.9ml59.4µl180 ml

2.4ml1.2ml79.2µl240 ml

3.0ml1.5ml300 ml

3.6ml 4.2ml1.8ml 2.1ml360 ml 420 ml

38.16ml57.24ml76.32ml95.4ml114.48ml 133.56ml99µl 118.8µl 138.6µl