A.rehydration/sample lysis buffer

(Ⅰ) 1ml

urea	8M	0.48g
CHAPS	4%	40mg
DTT	50~65mM	7~9.8mg
or TBP	>2mM	10µl
40% Bio-Lyte	>0.2%(w/v)	5µl
1% Bromphenol blue	>0.001%	1µl
MilliQ water	>	650µl

(Ⅱ) 1ml

urea	7M	0.42g
thiurea	2M	0.152g
CHAPS	4%	40mg
DTT	>50~65mM	7~9.8mg
or TBP	>2mM	10µl
40% Bio-Lyte	>0.2%(w/v)	5µl
1% Bromphenol blue	>0.001%	1µl
MilliQ water	>	650µl

1.(Ⅰ)The urea concentration in can be adjusted to 9 or 9.8m,The ability of dissolving protein with 7 murea and 2 m thoiurea is better than that with urea alone.
2.At present, the commonly used detergent is chaps, which can also be replaced by Triton X-100, NP-40, etc.
3.Protein enzyme inhibitors and / or nuclease can be added.
4.The reductant can be DTT or TBP, and TBP is the best way to separate basic protein.
5.Bromophenol blue can be used as indicator to monitor the sample loading and focusing process. If the operation is skilled, it can be dispensed.

B.Bromophenol blue reservoir

	Final concentration	Amount
Bromophenol blue	1%	10mg
Tris-base	50mM	6mg
MilliQ H2O	>	to 1ml

C.SDS equlibration buffer
(50mM Tris-cl pH8.8, 6M Urea, 30% glycerol, 2% SDS, bromophenol blue, 200ml)

	Final concentration	Amount
1.5M Tris-cl,pH8.8	50mM	6.7ml
Urea (Fw 60.06)	6M	72.07g
Glycerol(87% v/v)	>30%(v/v)	69ml
SDS(Fw 288.38)	>2%(w/v)	4.0g
1%Bromophenol blue solution	>0.002%(w/v)	400µl To 200ml

It can be stored at - 20 ℃; bromophenol blue can not be added;Add DTT (20mg / ml) or iodoacetamide (25mg / ml) before use.

D.10%(v/v)SDS solution

SDS(Fw 288.38)	10.0g
MilliQ H2O	To 100 ml

Use 0.45 μ M filter paper to filter and store at room temperature (high-purity reagent does not need to be filtered generally).

E.Monomer stock solution

	Final concentration	Amount
N,N ’ -methylenebisacrylamide or PDA	0.8%	4.0g 5.0g
MilliQ H2O		To 500 ml

Filter with 0.45 μM filter paper (not filtered), and store in dark at 4 ℃.

F.4x Resolving gel buffer, 1.5MTris-Cl pH8.8

	Final concentration	Amount
Tris-base(Fw 121.1) MilliQ H2O HCl(Fw 36.46)	1.5M	181.5g 750ml Adjust to pH8.8
MilliQ H2O		To 1000 ml

Filter with 0.45 μM filter paper and store at 4 ℃.

G.10% Ammonium persulfate

	Final concentration	Amount
Ammonium persulphate(APS) MilliQ H2O	10%	1g To 10ml

When water is added, the fresh ammonium persulfate makes a "click" sound. If there is no sound, a new medicine is needed. It can be prepared in small amount (10ml) and stored at 4 ℃ after subpackage.

H.TGS electophoresis buffer
(25mM Tris,192 mM glycine, 0.1%SDS)

	Final concentration	Amount
Tris base	25 mM	15.1g 7.55g
Glycine	192mM	72.1g 36.05g
SDS	0.1%(w/v)	5.0g 2.5g
MilliQ H2O		To 5000ml To 2500ml

The pH value of the solution does not need to be adjusted. It can be directly prepared in a large reagent bottle and stored at room temperature.

J.Agrose sealing solution
(25mM Tris,192 mM glycine, 0.1%SDS)

	Final concentration	Amount
TGS electophoresis buffer	---	50ml
Agrose (NA or M)	0.5%	0.25g
1% Bromophenol blue	0.002%(w/v)	100μl

It can be made in conical flask and melted in microwave oven. In small parts, store at room temperature.