Testing new anti-ORF2 beads and ORF2 dipeptides
Date: 05/27/21
Cell line: LD401 Light (05/02/14)
Extraction Buffer: 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 (v/v)
Scale: 50mg for LD401 anti-ORF2 IP (5ul of anti-ORF2 beads per 50mg IP)
Weigh out 2x 100mg of LD401 powder; add 400ul of extraction buffer (1:4 w:v)
Vortex to mix; put the tubes on ice after resuspending the powder
Sonicate 2 Amp, 4x 2 sec
After sonication, spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the clarified lysate
Set up the following 4x 50mg IP reactions:
1) anti-ORF2 (9-7)_011421 (two replicates)
2) anti-ORF2 (5-5)_052621 (two replicates)
IP @ 4°C for 30’ with rotation (cold room)
Wash beads with 3x 1ml extraction buffer
Transfer the beads to fresh tubes during 2nd wash
After the 3rd wash, spin down briefly and remove any remaining liquid
Native elution with ORF2 di-peptides from 21st Century
Prepare ORF2 di-peptide stocks @ 2.5mM
50mM HEPEs, pH 7.4, 500mM NaCl, 0.5% Triton X-100
Ac-KASRRQEITKIRAE-PEG4-KASRRQEITKIRAE-amide (ORF2, MT9)
MT9 dipeptide @ 2.5mM (average mass 3642Da)
Take one vial 2mg aliquots (purity >99%)
2.5M => 2.5x 3642mg/ml; 2.5mM => 2.5x 3642ug/ml=9.105mg/ml (To make 2.5mM stock, 2mg should be dissolved in 0.220ml final vol.)
Ac-QDIGVGKD-PEG4-QDIGVGKD-amide (ORF2, MT5)
MT5 dipeptide @ 2.5mM (average mass 1932Da)
Take one vial 2mg aliquots (purity >99%)
2.5M => 2.5x 1932mg/ml; 2.5mM => 2.5x1932ug/ml=4.83mg/ml (To make 2.5mM stock, 2mg should be dissolved in 0.414ml final vol.)
Dilute ORF2 di-peptide to 1mM with extraction buffer
Native elution with 5ul ORF2 dipeptide @ 2.5mM or 1mM
1) anti-ORF2 (9-7)_1mM MT9
2) anti-ORF2 (9-7)_2.5mM MT9
3) anti-ORF2 (5-5)_1mM MT5
4) anti-ORF2 (5-5)_2.5mM MT5
Incubate beads with peptide @ RT for 15’
Collect the elution (“E”)
Wash the beads in each tube with 5ul of extraction buffer
Combine wash with elution from the same tub (10ul total)
LDS wash of the beads after native elution
Wash beads with 10ul of 1.1x LDS for 10 min at RT w/ mixing
Transfer the LDS wash to a fresh tube (“LDS”)
Add 1x LDS and 50mM DTT (final concentrations) to gel samples as needed
Heat samples @ 70°C for 10’
Run 4-12% Bis-Tris gel
1) Marker
2) E_anti-ORF2 (9-7)_1mM MT9
3) E_anti-ORF2 (9-7)_2.5mM MT9
4) E_anti-ORF2 (5-5)_1mM MT5
5) E_anti-ORF2 (5-5)_2.5mM MT5
6) Space
7) LDS_anti-ORF2 (9-7)_1mM MT9
8) LDS_anti-ORF2 (9-7)_2.5mM MT9
9) LDS_anti-ORF2 (5-5)_1mM MT5
10) LDS_anti-ORF2 (5-5)_2.5mM MT5
11) Space
12) BSA_50ng
13) BSA_150ng
