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Lysis buffer

NP-40是一种商品化的去污剂，可以裂解细胞膜而不能裂解核膜，用它处理样品后可以获得胞质的内含物。
NP-40 is a commercially available detergent. This detergent is not powerful enough to break the nuclear membrane, but can break the cytoplasmic membrane. As such, it can be used to obtain the cytoplasmic contents of a cellular culture.

RIPA buffer——Radio Immuno Precipitation Assay buffer,RIPA裂解液可以用于全细胞和膜结合蛋白的抽提，在核蛋白抽提方面要优于单独使用NP-40或者 Triton X 100裂解液。但它会破坏蛋白-蛋白之间的作用力，故而不适用于IP或者pull down实验。

*10%的脱氧胆酸钠储备液必须避光保存。

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常用的缓冲液和储备液的配方

STOCK SOLUTION RECIPIES:

Tris-HCl Buffer

10X Tris-HCl (0.5M Tris Base, pH7.6):

Trizma Base ---------------------------------- 61 g

Distilled water ------------------------------- 1000 ml

Adjust pH 7.6 using concentrated HCl

Store this solution at room temperature. Dilute 1:10 with distilled water before use

and adjust pH if necessary.

20X Tris-HCl (1M Tris Base, pH7.6):

Trizma Base ---------------------------------- 122 g

Distilled water ------------------------------- 1000 ml

Adjust pH 7.6 using concentrated HCl

Store this solution at room temperature. Dilute 1:20 with distilled water before use

and adjust pH if necessary.

10X Tris-HCl-Tween 20 (0.5M Tris Base, 0.5% Tween 20, pH7.6):

Trizma Base ---------------------------------- 61 g

Distilled water ------------------------------- 1000 ml

Adjust pH 7.6 using concentrated HCl and then add 5 ml of Tween 20.

Store this solution at room temperature. Dilute 1:10 with distilled water before use

and adjust pH if necessary.

Note: Tris-HCl Buffer is used for specific cases of immunohistochemical staining.

*** OR you can use Tris Base to make Tris-HCl (note that Tris base is different

from Trizma)

Tris is a chemical with basic properties, having a pKa of 8.1. It can be used to buffer

solutions from drastic pH changes, keeping them in the pH range of 7.0 to 9.0.

Make any Tris-HCl buffer in this pH range, at any molarity using these simple steps

1) Calculate Moles of Tris Base

mol/L * L = moles needed

2) Calculate Mass of Tris Base

Determine the mass of Tris base to weigh by multiplying the number of moles by

the molecular weight (121.14 g/mol) of Tris.

moles needed * g/mol = g

3) Dissolve Tris Base in Water

Dissolve the required mass of Tris into a volume of deionized water

approximately 1/3 of the desired volume of buffer to be made.

4) Adjust the pH

Using a pH meter, titrate the solution of Tris with 1M hydrochloric acid (HCl)

until the correct pH is reached.

5) Bring to Volume

Add the TrisHCl mixture to a volumetric flask of the desired volume and add

deionized water as required to complete the solution.

0.2M Phosphate Buffer-4 Liters (pH 7.4):

17.66g Sodium Phosphate Monobasic

90.03g Sodium Phosphate Dibasic Heptahydrate

4 Liters ddH2O

pH should be 7.4, if not adjust with 1.0N NaOH or 1.0N HCl

(3 Liters-13.25g Mono and 67.52g Dibasic)

0.1M Phosphate Buffer Saline (PBS)-8 Liters:

Prepare 4 liters of 0.2M phosphate buffer (see above recipe)

Add 72g NaCl (0.9% or 9g/liter)

Add 4 liters of ddH2O

pH=7.4

*For practical purposes, you can also make 16 liters of PBS by first preparing 4 liters of

0.4M Phosphate Buffer. This concentration uses twice as much Monobasic and Dibasic

since 0.4M versus 0.2M means the solution is twice as concentrated. But remember, you

must then dilute this solution by adding 12 liters of water to make a total of 16 liters at a

concentration of 0.1M. Also, since we are using 9gNaCl/liter, a total of 144g of NaCl

will be used.

0.1M Phosphate Buffer-1 liter:

0.5 Liter of 0.2 M Phophate Buffer Stock

0.5 Liter of ddH2O

0.1M Phosphate Buffered Saline with Azide-1 liter (PBS*):

Prepare one liter of 0.1M Phosphate Buffer (see recipe above)

Add 9g of NaCl

Add 200mg of sodium azide (contents of 1 eppendorf vial)

Add 0.3 ml Triton-X

*Be sure to use protective clothing and a mask when handling sodium azide under hood!!

1% Paraformaldehyde 2% Glutaraldehyde

0.2M Stock Phosphate Buffer pH 7.4 500ml

Paraformaldehyde 10g

Glutaraldehyde (25% in water) 80ml

Distilledwater QS to 1000ml

Dissolve the paraformaldehyde in about 400 ml of water. Heat this solution to 58 to 60

degrees C(do not allow the solution to get to hot!!). Add 1N NaOH drop by dropuntil the

solution turns clear. Add the phosphate buffer stock and allow to cool, and then add the

glutaraldehyde. Ph the solution to 7.4 and FILTER before use.

1% Paraformaldehyde 2% Glutaraldehyde (Hrp Fix)

0.2M Stock Phosphate Buffer pH 7.4 500ml

Paraformaldehyde 10g

Glutaraldehyde (25% in water) 80ml

Distilled water QS to 1000ml

Dissolve the paraformaldehyde in about 400ml of water. Heat the solutionto 58 to 60

degrees C (do not allow the solution to get too hot!!). Add 1N NaOH drop by dropuntil

the solution clears.Add phosphate buffer stock and allow the solution to cool. Add

theglutaraldehyde and FILTER.

2% PARAFORMALDEHYDE

0.2M Stock Phosphate Buffer pH 7.4 500ml

Paraformaldehyde 20g

Distilled water QS to 1000ml

Dissolve the paraformaldehyde in about 400ml of water. Heat the solutionto 58 to 60

degrees C(do not allow the solution to get too hot!!). Add 1N NaOH drop by dropuntil

the solution clears.Add phophate buffer stock and allow the solution to cool. pH to 7.4

and FILTER.

4% PARAFORMALDEHYDE

0.2 M Stock Phosphate Buffer pH 7.4 500ml

Paraformaldehyde 40g

Distilled water QS to 1000ml

Dissolve the paraformaldehyde in about 400ml of water. Heat the solution to 58 to 60

degrees C(do not allow the solution to get too hot!!) Add 1N NaOH to the

paraformaldehyde solution drop by drop until the solution clears. Add phosphate buffer

stock and allow to cool. pH to 7.4 and FILTER.

4% PARAFORMALDEHYDE 0.2% GLUTARALDEHYDE

0.2M Stock Phosphate Buffer pH 7.4 500ml

Paraformaldehyde 40g

Glutaraldehyde (25% in water) 8ml

Distilled water QS to 1000ml

Dissolve the paraformaldehyde in about 400 ml of water. Heat the solutionto 58 to 60

C(do not allow the solution to get too hot!!). Add 1N NaOH drop by dropuntil the

solution clears. Add phosphate buffer stock and allow the solution to cool. Add the

glutaraldehyde. pH to 7.4 and FILTER.

Sucrose solution

10% 10gram in 90 ml 0.1 M PB

20% 20gram in 80 ml 0.1 M PB

30% 30gram in 70 ml 0.1 M PB

Acrylamide for separating gel (Acrylamide : BIS = 30 : 0.135)

Acrylamide 30.00 g

BIS 0.135 g

Make volume to 100 ml with MQ water. Keep in dark (Brown bottle)

Separating gel buffer (pH 8.8) (Final Conc.)

Tris 12.11 g 1 M

SDS 0.27 g 0.27%

Dissolve in 80 ml MQ water, adjust pH to 8.8, make the vol. to 100 ml

Acrylamide for stacking gel (Acrylamide : BIS = 29.2 : 0.8)

Acrylamide 29.2 g

BIS 0.8 g

Make volume to 100 ml with MQ water. Keep in dark (Brown bottle)

Stacking gel buffer (pH 6.8) (Final Conc.)

Tris 3.03 0.25 M

SDS 0.20 g 0.2%

Dissolve in 80 ml MQ water, adjust pH to 6.8, make the vol. to 100 ml

SDS-PAGE running buffer

Tris 9.0 g

Glycine 43.2 g

SDS 3.0 g

Dissolve in 3 L MQ water

SDS-sample buffer

Glycerol 10 ml

Tris 0.757 g

SDS 2.5 g

2-Mercaptoethanol 5.0 ml

Dissolve in 100 ml MQ water

Bromophenol blue (BPB) solution

Dissolve 0.1 g BPB in 100 ml 10% glycerol

Blotting solution A (Final conc.)

Tris 36.33 g 0.3 M

Methanol 200 ml 20%

SDS 0.20 g 0.02%

Make vol to 1000 ml with MQ water, keep at 4 ºC

Blotting solution B (Final conc.)

Tris 3.03 g 25 mM

Methanol 200 ml 20%

SDS 0.20 g 0.02%

Make vol to 1000 ml with MQ water, keep at 4 ºC

Blotting solution C (Final conc.)

Tris 3.03 g 25 mM

s-Amino-n-Caproic Acid 5.20 g 40 mM

Methanol 200 ml 20%

SDS 0.20 g 0.02%

Make vol to 1000 ml with MQ water, keep at 4 ºC

Ponceau 3S staining solution

Dissolve 0.1 g Ponceau 3S in 100 ml 1% Acetic acid

Coomassie brilliant blue (CBB) solution

Methanol 1500 ml

Acetic acid 300 ml

Coomassie brilliant blue-R250 3 g

Dissolve in 3000 ml MQ water

Destaining solution

Methanol 1100 ml

Acetic acid 300 ml

Dissolve in 3000 ml MQ water

Acrylamide solution for 1D-PAGE(Acrylamide : BIS = 28.38 : 1.62)

Acrylamide(High grade) 28.38 g

BIS 1.62 g

Make vol to 1000 ml with MQ water, keep in dark

 

Lysis buffer for Western Blot

Lysis buffer stock

1 ml Tris HCl, pH 7.4 (pre-made stock)

0.584g NaCl

49 ml dH2O

This stock can be kept for up to 2 weeks at 4C

Lysis buffer for tissue prep

10 ml lysis buffer stock (see above)

1 protease inhibitor tablet

0.2 ml Phosphatase inhibitors (1 mM Na3VO4 and 5 mM NaF to block both tyrosine

and serine/threonine kinases, see below)

100 ml (50x Na3VO4 and NaF stock solution)

50 mM Na3VO4 0.92gl

500 mM NaF 2.0995g

100 ml dH2O

0.02 N H3PO4

Add 0.48 ml H3PO4 (42 N, specific weight 1.87, 72%) to 1000 ml MQ water

0.02 N NaOH

Dissolve 0.8 g NaOH to 1000 ml MQ water.

www.abcam.com/technical

RIPA buffer (RadioImmunoPrecipitation Assay) buffer:

RIPA buffer contains the ionic detergent sodium deoxycholate as an active constituent

and is particularly used for nuclear membrane disruption for nuclear extracts. A RIPA

buffer gives low background but can denature kinases. It can also disrupt protein-protein

interactions (and may therefore be problematic for

immunoprecipitations/pull down assays).

50mM Tris HCl pH 8

150 mM NaCl

1% NP-40

0.5% sodium Deoxycholate

0.1% SDS

The 10% sodium deoxycholate stock solution (5 g into 50 ml) must be protected from

light.

The 100 mM EDTA stock solution is made with 1.86 g into 40 ml H2O and then add

NaOH to dissolve and adjust pH to 7.4. Finally, adjust the total volume to 50 ml). Store

the buffer at 4°C.

Nonidet-P40 (NP-40) buffer:

20 mM Tris HCl pH 8

137 mM NaCl

10% glycerol

1% nonidet P-40

2 mM EDTA

Sodium orthovanadate preparation:

This needs to be done under the fume hood

• Prepare a 100 mM solution in double distilled water

• Set pH to 9.0 with HCl

• Boil until colorless

• Cool to room temperature

• Set pH to 9.0 again

• Boil again until colorless

• Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling

• Bring up to the initial volume with water

• Store in aliquots at -20°C

Note: do not permit great changes in volume during boiling; put a loose lid on the

container to protect from evaporation. Discard if the samples turn yellow.

TBS 10x (concentrated TBS)

24.23 g Trizma HCl

80.06 g NaCl

Mix in 800 ml ultra pure water.

pH to 7.6 with pure HCl.

Top up to 1 L.

TBST

For 1 L:

100 ml of TBS 10x

+ 900 ml ultra pure water

+ 1ml Tween20

Medium stripping buffer:

Make fresh stripping buffer:

15 g glycine

1 g SDS

10 ml Tween20

Set the pH to 2.2

make up to 1 L with ultrapure water

Harsh stripping buffer:

to be done under the fumehood

For 100 ml:

20 ml SDS 10%

12.5 ml Tris HCl pH 6.8 0.5M

67.5 ml ultra pure water

Add 0.8ml ß-mercaptoethanol under the fumehood.

Nuclear Fractionation Protocol Reagents

Buffer A –

10 mM HEPES,

1.5 mM MgCl2,

10 mM KCl,

0.5 mM DTT,

0.05% NP40 (or 0.05% Igepal or Tergitol)

pH 7.9

To prepare 250 ml stock of buffer A –

HEPES: 1M = 238.3 g/L, therefore 10 mM = 0.59 g/250 ml

MgCl2: 1M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 ml

KCl: 1M = 74.5 g/L, therefore 10 mM = 0.187 g/250 ml

DTT: 1M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 ml

NP40 = 0.05%

Buffer B –

5 mM HEPES,

1.5 mM MgCl2,

0.2 mM EDTA,

0.5 mM DTT,

26% glycerol (v/v)

pH 7.9

To prepare 250 ml stock of buffer B –

HEPES: 1M = 238.3 g/L, therefore 5 mM = 0.295 g/250 ml

MgCl2: 1M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 ml

EDTA: 1M = 372.2 g/L, therefore 0.2 mM = 0.0186 g/250 ml

DTT: 1M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 ml

26% Glycerol (v/v) = 65 ml

4.6 M NaCl

87.66 g/326 ml

TBS (Tris Buffered Saline) pH 7.6-7.8:

For 10 litres:

60.6 g TRIS HCl

13.9 g TRIS base

87.66 g NaCl

10 litres Ultra pure water (H2O)

TBS 0.025% Triton X-100:

For 1 litre:

250 μl Triton X-100

999.75 ml TBS

pH 7.6-7.8

1.6% H2O2 (Hydrogen Peroxide) in TBS:

For 400 ml:

6.4 ml H2O2 (GPR = 30% w/w)

393.6 ml TBS

pH 7.6-7.8

BS - Blocking serum in TBS (100ml):

NGS (10%) 10ml

BSA (2%) 2ml

Triton (0.4%) 4ml of 10% Triton

10% NS (Normal Serum) with 1% BSA (Bovine Serum Albumin, Fraction 5) in

TBS:

For 1 ml:

100 μl NS

10 mg BSA

900 μl TBS

pH 7.6-7.8

Primary antibody made up in TBS with 1% BSA:

(Example is of primary antibody used at a dilution of 1:10)

For 0.1 ml:

100 μl Primary antibody

10 mg BSA

900 μl TBS

pH 7.6-7.8

Secondary biotinylated antibody made up in TBS with 1% BSA:

(Example is of secondary biotinylated antibody used at a dilution of 1:200)

For 1 ml:

5 μl Secondary biotinylated antibody

995 μl TBS

pH 7.6-7.8

ABC (Avidin-Biotin) complex in TBS:

(Example is of ABC complex, each part used at a dilution of 1:100)

For 1 ml:

10 μl Streptavidin

10 μl HRP (or AP)-Biotin

980 μl TBS

pH 7.6-7.8

Bicarbonate/carbonate coating buffer (100 mM):

3.03 g Na2CO3,

6.0 g NaHCO3 (1 L distilled water)

pH 9.6,

PBS (500 ml):

1.16 g Na2HPO4,

0.1 g KCl,

0.1 g K3PO4,

4 g NaCl

(500 ml distilled water) pH 7.4