硫酸盐沉淀
OUTLINE Ammonium sulphate precipitation is used to purify a protein (in this case an immunoglobulin) from a big volume of liquid phase.
PROTOCOL
Slowly (!!!) add the solution of ammonium sulphate (40-50% final, v/v) to hybridoma cell culture supernatant (60-50% final, v/v). Store at 4ѓC ,ON (overnight). centrifuge the precipitant at 5000-10000 rpm for 10-15 min at RT. resuspend the precipitant in PBS (as small volume as best). dialyse against PBS, ON for 48 hours. filter on 0.22 µm filter. run ELISA to establish the Ig concentration
SOLUTIONS
ammonium sulphate, saturated, pH 6.8, store at RT PBS
| ѓ |
[ p u r i f i c a t i o n o n p r o t e i n G ] OUTLINE Protein A (from Staph. aureus) and protein G (from Streptococcus sp. [Lancefield Group G]), both exhibit an affinity for the Fc portion of diverse array immunoglobulins from many species. Protein A binds to Fc-gamma and Fv-VHIII . Protein G binds to Fc-gamma and c-gamma1 chains.
PROTOCOL
| Step | Description | Details |
| Step 0 - Delipidtion | Use this step when purifying ascites or serum Use SeroClear : ascitesFluid/serum = 1 : 1,5 ->vortex 1 min,RT->spin 5000 rpm ,10 min-> retain the top layer for subsequent purification | <!--[endif]--> SeroClear- CalBiochem cat. No .437616 , not compatible with polysterene tubes 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 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. |
| Step 1 - Equilibration & Loading | Equilibrate the column with 5 - 10 CV(column volums) of 20 mM sodium phosphate , pH 7 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 Loading rate : 50 ml/hr ( 0,8 ml/min) | 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 alternatively it`s possible to incubate the gel directly with serum or ascitesFluid for 30 min before applying the gel to the column alternatively dilute 2 -fold for tissue culture SN or 10 -fold for ascitesFluid and bioreactor SN in the loading buffer |
| Step 2 - Whashing | wash the column with 20 mM sodium phosphate , pH 7 collect the flow-through | usually 5 - 10 CV are used to wash the column check the OD 280 of the flow through to determine when whashing is complete |
| Step 3 - Elution | elute the bound Abs with 0.1 M glycine-HCl , pH 2.7 ( 2.2 ) collect fractions and monitor the OD 280 collect fractions into tubes containing 50 - 100 ul 2 M TRIS base , pH 8.0 per 1 ml of fraction , pool fractions | alternatively use 0,1 M acetic acid , pH 2.8 to elute 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 Add 0,05 % NaN 3 if stored at 2 - 8 C and 50 % glycerol for - 20 C |
| Step 4 -Washing, Cleaning and Gel storage , Re-equilibration | Wash with 0.1 M glycine-HCl , pH 2.7 Clean with C 2 H 5 OH 70 % -wash , incubate for 12 hrs Store with 20 % C 2 H 5 OH, store at 2 - 8 C, don`t freeze Re-equilibration with 20 mM sodium phosphate , pH 7 | alternatively use 1 M acetic acid for washing ( 3 - 4 CV) alternatively use 100 mM PBS , pH 7.4 with Proclin 0.01 % as a preservative for storage alternatively use 0.1 M sodium acetate , pH 5.0 for re-equilibration |
SOLUTIONS
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 |
相关文章
