Conjugation of anti-FLAG Dynabeads
Date: 09/02/20
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
RU beads: lot # 00816517 (exp date 2021-02-28)
ERIBA beads: lot # 00734116 (exp date 2020-04-30)
Check the concentration by Bradford Assay and use desalted antibody to make beads
Anti-FLAG beads
Anti-FLAG (M2, Sigma F3165 in PBS, use it directly without desalting), use 10ug of antibody per mg of beads. The antibody concentrationis 3.71mg/ml. Will also make 75mg of beads total, 25mg of ERIBA beads and 50mg of RU beads. Total antibody needed will be 0.75mg (200ul).
Anti-FLAG from Marty
Mouse anti-FLAG, either desalted into PBS or gel filtered into PBS.
If desalted it will be 13.3 mg/ml (A280=18) and 750 ul (This is what we got)
If SEC purified it will be 13.9mg/ml (A280=18.9) and 720 ul
Antibody mix: 10ug of anti-FLAG antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads
Take 2ul of antibody mix before coupling for gel analysis
Add antibody mix to beads pre-equilibrated with 0.1M NaPO4, pH7.4; mix well
Leave the tube on a Thermomixer at 37°C (1100rpm), O.N. (18 - 24hr)
Take 2ul of antibody mix after coupling for gel analysis
Keep the rest of the antibody mix @ 4°C
Wash the beads 1x 1ml 100mM Glycine-HCl, pH 2.5
Wash the beads 1x 1ml 10mM Tris, pH 8.8
Wash the beads 1x 1ml fresh 100mM Triethylamine
Wash the beads 4x 1ml 1x PBS (5’ each)
Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 5’
Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 15’
Resuspend beads in 50% Glycerol/1xPBS/0.5mg/ml BSA for long-term storage at -20°C
Check antibody mix before and after beads conjugation on 4-12% Bis-Tris gel
Loading:Marker->antibody mix before and after conjugation (1-7)
Orf2 (MT302) PO testing anti-FLAG beads
Date: 09/04/20
Cell lines: MT302 Light, 05/02/14
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 25mg per IP reaction with 5ul of anti-FLAG beads
Beads: 10ug per mg of beads unless it is specified otherwise; anti-FLAG antibody is from Marty; Sigma M2 anti-FLAG is called F3165 here
1) anti-FLAG_E_E_090320 (ERIBA beads_ERIBA buffer)
2) anti-FLAG_E_R_090320 (ERIBA beads_RU buffer)
3) anti-FLAG_R_E_090320 (RU beads_ERIBA buffer)
4) anti-FLAG_R_R_090320 (RU beads_RU buffer)
5) anti-FLAG_R_R_15_090320 (RU beads_RU buffer, 15ug of anti-FLAG per mg of beads)
6) anti-FLAG_R_R_20_090320 (RU beads_RU buffer, 20ug of anti-FLAG per mg of beads)
7) F3165_R_R_090320
8) anti-FLAG_ERIBA_060820 (from Leila)
9) anti-FLAG_RU_060820 (from Leila)
10) F3165_ERIBA_060820 (from Leila)
11) F3165_RU_060820 (from Leila)
Anti-ORF1 IP
Weigh out 2x 137.5mg of MT302 (275mg total), add 550ul of extraction buffer to each tube (1:4 w/v), vortex to mix
New sonicator: Sonicate 5 x 2 sec at 2 Amp;
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the supernatant, split it evenly into 6 aliquots
Set up 11x 25mg IP reactions with 5ul of beads
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute the beads with 10ul of 1.1x LDS, 70 °C for 5’ with mixing
Collect the eluate
Add DTT to 50mM to all samples and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze the elution, BlueSiver Stain
1) Marker
2) anti-FLAG_E_E_090320
3) anti-FLAG_E_R_090320
4) anti-FLAG_R_E_090320
5) anti-FLAG_R_R_090320
6) anti-FLAG_R_R_15_090320
7) anti-FLAG_R_R_20_090320
8) F3165_R_R_090320
9) anti-FLAG_ERIBA_060820
10) anti-FLAG_RU_060820
11) F3165_ERIBA_060820
12) F3165_RU_060820
13) BSA_50ng
14) BSA_150ng
DMP XL of anti-FLAG beads (Marty’s antibody)
Date: 09/09/20
Take 20ul of each kind of beads
Wash in 1ml of 1x PBS
Resuspend beads in 500ul of 20mM DMP in Borate buffer (~2ml per reaction with 100ul of beads; make this fresh)
Perform crosslink reaction at RT on Digital Tube Rotator for 30’.
After 30’, remove the crosslink solution
Wash beads with 1ml of PBST
Wash beads again with 1ml of TBST for 5’ at RT
Wash beads again with 1ml of PBST
Wash beads with 1ml of 1x PBS
Continue with wash steps in standard conjugation protocol
After all wash steps, resuspend beads in Magnetic Media Storage Solution and kept beads in -20°C freezer.
Take 2x 5ul of beads before XL, wash with 1x PBS, elute with 10ul of LDS @ 25C for 10’ or 70C of 5’
Take 5ul of beads after XL, wash with 1x PBS, elute with 10ul of LDS @ 70C of 5’
Run all LDS elution of gel
1) Marker
2) R_E_anti-FLAG_RT
3) R_E_anti-FLAG _70C
4) R_E_anti-FLAG+XL_70C
5) R_R_anti-FLAG_RT
6) R_R_anti-FLAG _70C
7) R_R_anti-FLAG+XL_70C
8) R_R_F3165_RT
9) R_R_F3165_70C
10) R_R_F3165+XL_70C
11) R_E_anti-FLAG_Leila_RT
12) R_E_anti-FLAG_Leila _70C
13) R_E_anti-FLAG_Leila +XL_70C
14) BSA_50ng
15) BSA_150ng
