硫酸盐沉淀

1. Use this step when purifying ascites or serum

    

2. Use SeroClear : ascitesFluid/serum = 1 : 1,5 ->vortex 1 min,RT->spin 5000 rpm ,10 min-> retain the top layer for subsequent purification

1. SeroClear- CalBiochem cat. No .437616 , not compatible with polysterene tubes

    

2. Alternatively mix 3 : 2 = 1 , 1 , 2 -trichlorotrifluoro -ethane:serum/ascitesFluid->shake 30 min,RT,spin 5000 rpm,RT ,10 min -> retain the top layer for subsequent purification
3. Alternatively for ascitesFluid use a glass wool by placing into a funnel to cover the opening, pouring ascites through, rinsing glass wool with PBS, and squeezing glass wool gently with gloved fingers to obtain all the sample. Centrifuge filtered ascites 30 min at 20000 g ,RT or 4 C. Decant and save the SN.

1. Equilibrate the column with 5 - 10 CV(column volums) of 20 mM sodium phosphate , pH 7

    

2. Dilute ascites or serum 1 : 1 with 20 mM sodium phosphate , pH 7 , !FILTER 0,8 -> 0,45 - 0,22 um and then load onto the column

    

3. Loading rate : 50 ml/hr ( 0,8 ml/min)

1. Alternatively it`s possible to use PBS x 1 instead of 20 mM sodium phosphate or 0.1 M sodium acetate , pH 5.0 or 10 mM sodium phosphate, pH 7.0 with 0.15 M NaCl for equilibration-loading-washing

    

2. alternatively it`s possible to incubate the gel directly with serum or ascitesFluid for 30 min before applying the gel to the column

    

3. alternatively dilute 2 -fold for tissue culture SN or 10 -fold for ascitesFluid and bioreactor SN in the loading buffer

1. wash the column with 20 mM sodium phosphate , pH 7

2. collect the flow-through

1. usually 5 - 10 CV are used to wash the column
2. check the OD 280 of the flow through to determine when whashing is complete

1. elute the bound Abs with 0.1 M glycine-HCl ,      pH 2.7 ( 2.2 )

    

2. collect fractions and monitor the OD 280

    

3. collect fractions into tubes containing 50 - 100 ul 2 M TRIS base , pH 8.0 per 1 ml of fraction , pool fractions

1. alternatively use 0,1 M acetic acid , pH 2.8 to elute

    

2. Ab can be stored in 2 M TRIS base , pH 8.0 ,or can be exchanged for PBS x 1 or TRIS buffer by dialysis

    

3. Add 0,05 % NaN 3 if stored at 2 - 8 C and 50 % glycerol for - 20 C

1. Wash with 0.1 M glycine-HCl , pH 2.7

    

2. Clean with C 2 H 5 OH 70 % -wash , incubate for 12 hrs
3. Store with 20 % C 2 H 5 OH, store at 2 - 8 C, don`t freeze     
4. Re-equilibration with 20 mM sodium phosphate , pH 7

1. alternatively use 1 M acetic acid for washing ( 3 - 4 CV)

    

2. alternatively use 100 mM PBS , pH 7.4 with Proclin 0.01 % as a preservative for storage

    

3. alternatively use 0.1 M sodium acetate , pH 5.0 for re-equilibration

Buffer

Components & details

20 mM sodium phosphate, pH 7.0

3,27 g Na 2 HPO 4 x 7 H 2 O

0,94 g NaH 2 PO 4

q.s. ddH 2 O to 1 L

correct pH to 7.0

! Use NaOH or HCl to adjust pH being careful not to overshoot and back-titrate, as this may alter salt concentration more than necessary

10 mM sodium phosphate, pH 7.0 with 0.15 M NaCl

1.64 g NaH 2 PO 4 x 7 H 2 O

0.47