Testing new anti-ORF2 (clone 9-7) beads
Conjugation of anti-ORF2 beads (08/17/21)
Anti-ORF2 beads
Anti-ORF2 antibody (clone 9-7) from abcam, 1mg/ml in 1x PBS, pH7.4 with 0.01% azide, can be directly used for conjugation; use 0.5mg of antibody to make 50mg of beads (beads are from RU)
500ul anti-ORF2 antibody (10ug Ab / mg beads,0.5mg antibody)
333ul 3M AmSO4 (final concentration 1M)
167ul 0.1M NaPO4, pH7.4
Compare Ab mix before and after coupling to see reduction of antibody (see last two lanes on sypro gel before Western section)
Testing new anti-ORF2 (clone 9-7) beads (08/18/21)
Cell line: LD401 heavy (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 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 2x 50mg IP reactions:
1) anti-ORF2 (9-7)_011421 (old batch of clone 9-7, received in 2019)
2) anti-ORF2 (9-7)_081821 (new batch of clone 9-7, received in 2020)
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; working concentration 1mM
Peptide buffer: 50mM HEPEs, pH 7.4, 500mM NaCl, 0.5% Triton X-100
Dilute ORF2 di-peptide to 1mM with extraction buffer
Native elution with 5ul ORF2 dipeptide @ 1mM
1) E_anti-ORF2 (old 9-7)_MT9_RT 15’
2) E_anti-ORF2 (new 9-7)_MT9_RT 15’
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 two 4-12% Bis-Tris gels (load 90% of each sample on one gel for Sypro stain; save10% of each sample for Western)
1) Marker_1ul unstained
2) E_anti-ORF2 (old 9-7)_MT9_RT 15’
3) E_anti-ORF2 (new 9-7)_MT9_RT 15’
4) LDS_anti-ORF2 (old 9-7)
5) LDS_anti-ORF2 (new 9-7)
6) Space
7) BSA_50ng
8) BSA_100ng
9) Space
10) Marker_1ul unstained
11) Anti-ORF2 Ab mix before coupling_2ul
12) Anti-ORF2 Ab mix before coupling_2ul
Sypro gel
2 sec exposure
Western
Loading order
1) Marker_5ul prestained
2) E_anti-ORF2 (old 9-7)_MT9_RT 15’
3) E_anti-ORF2 (new 9-7)_MT9_RT 15’
4) LDS_anti-ORF2 (old 9-7)
5) LDS_anti-ORF2 (new 9-7)
6) Space
7) E_10% of 50mg MT302 anti-ORF1 IP
8) Marker_2.5ul prestained
Wet transfer, 70V for 2h
Block the membranes in TBST/5% milk @ RT, 2hr
Cut the membrane in half, between 75 and 50KD
Upper panel: Rabbit anti-ORF2 Western (Clone 11)
Primary Ab: Rabbit anti-ORF2 antibody (Clone 11, check concentration), 1:500, 4°C, overnight
Secondary Ab: ECL anti-rabbit HRP 1:5,000, RT, 1hr
Lower panel: Anti-ORF1 Western (Abmart, 4H1, 2mg/ml)
Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
30 sec exposure, high sensitivity, auto tone
