硫酸盐沉淀
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
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[ 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 |
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Step 1 - Equilibration & Loading |
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Step 2 - Whashing |
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Step 3 - Elution |
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Step 4 -Washing, Cleaning and Gel storage , Re-equilibration |
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SOLUTIONS
Buffer
| Components & details
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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
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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 |