Recipes for buffers

RBC Lysis Buffer,          TBST,                  NP-40 Cell Lysis Buffer,                             Proteinase K Solution,     

RIPA buffer,                    Modified RIPA buffer,                                  ELISA coating buffer

10X Red Blood Cell (RBC) Lysis buffer


    90g NH4Cl  (0.1555M)

    10g KHCO3  (0.01M)

    370mg  EDTA  (0.1mM)


Or  1X RBC lysis buffer:

To 800ml dH2O add:

         8.3g ammonium chloride (NH4Cl )

         1.0g potassium bicarbonate (KHCO3)

         1.8ml of 5% EDTA 


Filter sterilize through 0.2um filter.

Bring to final volume of 1000ml with dH2O.

 

 

TBST

(50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.4)

For 1 liter of 1X Tris-buffered saline with Tween-20 (1X TBST buffer)

-Dissolve in 800ml of distilled H2O:

8g NaCl

0.2g KCl

3g Tris base

-Add 500ul Tween-20

-Adjust the pH to 7.4 with HCl

-Add distilled H2O to 1L


For 1 liter of 10X Tris-buffered saline with Tween-20 (10X TBST buffer)

-Dissolve in 800ml of distilled H2O:

80g NaCl

20g KCl

30g Tris base

-Add 500ul Tween-20

-Adjust the pH to 7.4 with HCl

-Add distilled H2O to 1L

NP-40 Cell Lysis Buffer

50mM Tris-HCl pH 8.0

150mM NaCl

1% NP-40

plus protease inhibitors: Aprotinin, leupeptin, pepstatin: 1ug/ml each

Add 1mM PMSF immediately before use.

See: P9070, Protease Inhibitor Cocktail

 

Proteinase K Solution

(20 ug/ml in TE Buffer, pH 8.0):

TE Buffer (50mM Tris Base, 1mM EDTA, 0.5% Triton X-100, pH 8.0):

Tris Base -------------------------------- 6.10 g

EDTA ------------------------------------- 0.37 g


Triton X-100 ---------------------------- 5 ml

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

Mix to dissolve. Adjust pH 8.0 using concentrated HCl (10N HCl). Store at room temperature.

 

RIPA buffer

50mM Tris, pH 8.0

150mM sodium chloride

0.1% SDS


0.5% Sodium deoxycholate

1% NP40

plus protease inhibitors: Aprotinin, leupeptin, pepstatin: 1ug/ml each

Add 1mM PMSF immediately before use.

See: R2031-75, RIPA Lysis Buffer

P9070 , Protease Inhibitor Cocktail

Modified RIPA buffer

10 mM Tris, pH 7.4

100 mM NaCl

1 mM EDTA

1 mM EGTA

1 mM NaF

20 mM Na4P2O7

2 mM Na3VO4

1% Triton X-100

10% glycerol

0.1% SDS

0.5% deoxycholate

plus protease inhibitors: Aprotinin, leupeptin, pepstatin: 1ug/ml each

Add 1mM PMSF immediately before use.

See: C2605-70, Cell Extraction Buffer

P9070, Protease Inhibitor Cocktail

 

ELISA coating buffer


To 900ml dH20, add:

5.3g of Na2CO3

4.2g of NaHCO3

1g sodium azide

Adjust pH to 9.6

Adjust volume to 1L with additional dH2O

Note: For coating step add 0.1-1ug/ml of coating antibody to buffer

and incubate at 4°C for 12-24 hours.