Testing new anti-ORF2 beads and ORF2 dipeptides_2nd round
Date: 06/03/21
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-ORF1 beads per 50mg IP)
Weigh out 4x 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 8x 50mg IP reactions: To save LD401 powder, time course for native elution will be done only with MT9 peptide
- anti-ORF2 (9-7)_011421 (5x)
- anti-ORF2 (5-5)_052621 (3x)
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
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 @ 1mM (positive control: 1mg/ml 3x FLAG; negative control: no peptide, incubate with peptide buffer)
1) anti-ORF2 (9-7)_buffer only_RT 30’ (negative control)
2) anti-ORF2 (9-7)_MT9_RT 15’
3) anti-ORF2 (9-7)_MT9_RT 30’
4) anti-ORF2 (9-7)_MT9_RT 60’
5) anti-ORF2 (9-7)_3x FLAG_RT 30’ (This was not a good control, should use anti-FLAG beads and 3xFLAG peptide as a pair)
6) anti-ORF2 (5-5)_buffer only_RT 30’ (negative control)
7) anti-ORF2 (5-5)_1mM MT5 RT 30’
8) anti-ORF2 (5-5)_3x FLAG_RT 30’ (This was not a good control, should use anti-FLAG beads and 3xFLAG peptide as a pair)
Incubate beads with peptide @ RT , incubation time see above
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 26-well 4-12% Bis-Tris gels (load 90% of each sample on one gel for Sypro stain; load 10% of each sample on the other gel for Western)
1) Marker (1ul unstained for Sypro gel; 5ul of prestained for Western)
2) E_anti-ORF2 (9-7)_buffer only_RT 30’ (negative control)
3) E_anti-ORF2 (9-7)_MT9_RT 15’
4) E_anti-ORF2 (9-7)_MT9_RT 30’
5) E_anti-ORF2 (9-7)_MT9_RT 60’
6) E_anti-ORF2 (9-7)_3x FLAG_RT 30’
7) E_anti-ORF2 (5-5)_buffer only_RT 30’ (negative control)
8) E_anti-ORF2 (5-5)_1mM MT5 RT 30’
9) E_anti-ORF2 (5-5)_3x FLAG_RT 30’
10) Space (1ul prestained marker was loaded for Western)
11) LDS_anti-ORF2 (9-7)_buffer only_RT 30’ (negative control)
12) LDS _anti-ORF2 (9-7)_MT9_RT 15’
13) LDS _anti-ORF2 (9-7)_MT9_RT 30’
14) LDS _anti-ORF2 (9-7)_MT9_RT 60’
15) LDS _anti-ORF2 (9-7)_3x FLAG_RT 30’
16) LDS _anti-ORF2 (5-5)_buffer only_RT 30’ (negative control)
17) LDS _anti-ORF2 (5-5)_1mM MT5 RT 30’
18) LDS _anti-ORF2 (5-5)_3x FLAG_RT 30’
19) Space
20) BSA_50ng (not on the gel for Western) will load 10% of 50mg MT302 anti-ORF1 IP here for Western
21) BSA_100ng (not on the gel for Western) will load 2.5ul of prestained marker for Western
Sypro gel
2 second exposure
Western
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, 0.445mg/ml), 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
1 min exposure, high sensitivity, auto tone
Conclusion: Clone 5-5 works very poorly for IP. MT5 did not work for native elution.