SEC of ORF 2 monoclonal antibody from John
Aim: Remove high molecular weight contaminants in his anti-ORF2 monoclonal antibody prep.
Materials:
Superose 200 increase 10/300 GL column (GE): 10kDa → 600 kDa separation range
buffer: 1 x PBS
Loaded 500 uL
Flow rate: 0.25 mL/min
Results
Fig.1 FPLC trace of anti-ORF2 monoclonal antibody separation
Fig 2: SDS-PAGE of SEC fractions
1. MW marker (precision plus all blue, Bio-Rad)
2. Space
3. Anti-ORF2 (2ul, before gel filtration)
4. Space
5. Fraction 10 (10ul)
6. Fraction 12 (10ul)
7. Fraction 13 (10ul)
8. Fraction 14 (10ul)
9. Fraction 15 (10ul)
10. Space
11. BSA_50ng
12. BSA_150ng
Fractions 12 - 1contain protein (peak fractions 13 and 14) = in agreement with FPLC trace. 3 bands? Usually there’s a heavy chain at 50 kDa and a light chain at 25 kDa.
BCA Assay to determine protein concentration is each fraction (500ul reaction volume, enhanced protocol, 60°C for 30’)
