硫酸盐沉淀

OUTLINEAmmonium 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

[ p u r i f i c a t i o n o n p r o t e i n G ]OUTLINEProtein 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
Step0- 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.
Step1- Equilibration & Loading	Equilibrate the column with 5 - 10 CV(column volums) of20mM sodium phosphate , pH7 Dilute ascites or serum 1 : 1 with20mM sodium phosphate , pH7, !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 usePBS x1instead of20mM sodium phosphate or0.1M sodium acetate , pH5.0or10mM sodium phosphate, pH7.0with0.15M NaClfor 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
Step2- Whashing	wash the column with20mM sodium phosphate , pH7 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
Step3- Elution	elute the bound Abs with0.1M glycine-HCl , pH2.7(2.2) collect fractions and monitor the OD280 collect fractions into tubes containing 50 - 100 ul2M TRIS base , pH8.0per 1 ml of fraction , pool fractions	alternativelyuse0,1M acetic acid , pH2.8to elute Ab can be stored in2M TRIS base , pH8.0,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
Step4-Washing, Cleaning and Gel storage , Re-equilibration	Washwith0.1M glycine-HCl , pH2.7 CleanwithC2H5OH70%-wash , incubate for 12 hrsStorewith20% C2H5OH,store at 2 - 8 C, don`t freeze Re-equilibrationwith20mM sodium phosphate , pH7	alternativelyuse1M acetic acidfor washing ( 3 - 4 CV) alternatively use100mMPBS , pH7.4with Proclin0.01%as a preservative for storage alternatively use0.1M sodium acetate , pH5.0for re-equilibration