Testing anti-FLAG and uncoupled beads from ERIBA
Dates: 07/07/20 - 07/15/20
Conjugation of anti-ORF1 and anti-FLAG Dynabeads
Date: 07/07/20
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-ORF1: Abmart (5310-1-4/4H1) received on 11/18/19; desalt into 0.1mM NaPhosphat, pH7.4 using Zeba 7k desalting column. Antibody concentration after desalting was 1.01mg/ml (total vol. 1.2ml). We will use 15ug of anti-ORF1 per mg of beads. We will have enough antibody to make 75 mg of beads. Use 25mg of beads from ERIBA and 50mg of beads from RU.
Antibody mix: 15ug of anti-ORF1 antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads (make antibody mix for 75 mg; use 1/3 for 25mg of beads, 2/3 for 50mg reaction)
1100 ul anti-ORF1 antibody
375 ul 4M AmSO4 (final concentration 1M)
25 ul 0.1M NaPO4, pH7.4
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).
Antibody mix: 10ug of anti-FLAG antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads (make antibody mix for 75 mg; use 1/3 for 25mg of beads, 2/3 for 50mg reaction)
200 ul anti-FLAG antibody (F3165, 3.71mg/ml)
500 ul 3M AmSO4 (final concentration 1M)
800 ul 0.1M NaPO4, pH7.4
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, 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
Orf2 (MT302) PO anti-ORF1 and anti-FLAG beads
Date: 07/08/20
Cell lines: MT302 Light, 05/02/14
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 50mg per IP reaction with 5ul of anti-ORF1 beads
Beads:
1) Old anti-FLAG beads from Leila
2) New anti-FLAG beads (uncoupled beads from ERIBA)
3) New anti-FLAG beads (uncoupled beads from RU)
4) Old anti-ORF1 Beads_060419
5) Old anti-ORF1 beads (uncoupled beads from ERIBA)
6) Old anti-ORF1 beads (uncoupled beads from RU)
anti-FLAG and anti-ORF1 IP
Weigh out 3x 100mg MT302, add 400ul 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 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) Old anti-FLAG beads from Leila
3) New anti-FLAG beads (uncoupled beads from ERIBA)
4) New anti-FLAG beads (uncoupled beads from RU)
5) Old anti-ORF1 Beads_060419
6) Old anti-ORF1 beads (uncoupled beads from ERIBA)
7) Old anti-ORF1 beads (uncoupled beads from RU)
8) Anti-FLAG_AbMix_PreCoupling
9) Anti-FLAG _AbMix_PostCoupling_ERIBA beads
10) Anti-FLAG _AbMix_PreCoupling_RU
11) Anti-ORF1_AbMix_PreCoupling
12) Anti-ORF1_AbMix_PostCoupling_ERIBA beads
13) Anti-ORF1_AbMix_PreCoupling_RU
14) BSA_50ng
15) BSA_150ng
Check LDS elution of anti-FLAG beads
Date: 07/11/20
Take 5ul of anti-FLAG beads from 3 different batches; Wash 1x with 1xPBS, elute with 10ul LDS with or without 50mM DTT at either 70C or 95C for 5’
1) Old beads from Leila (Sent from ERIBA, FedEx delivery delayed, beads were left at RT over the weekend by FedEx)
2) New beads made with Dynabeads from RU, kept at 4C
3) New beads made with Dynabeads sent from ERIBA, left at RT over the weekend
Collect the elution and add 50mM DTT to all samples with DTT already added during elution
Heat sample at 70C for 5’
Run on a 15-well 4-12% Bis-Tris gel, BlueSiver Stain
1) Marker
2) Old beads_70C_no DTT
3) Old beads_70C_with 50mM DTT
4) Old beads_95C_no DTT
5) New beads_RU_70C_no DTT
6) New beads_RU_95C_no DTT
7) New beads_ERIBA_70C_no DTT
8) New beads_ERIBA 95C_no DTT
9) BSA_50ng
10) BSA_150ng
Conjugation of anti-FLAG Dynabeads using anti-FLAG recovered from post-coupling antibody mix
Date: 07/14/20
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
ERIBA beads: lot # 00734116 (exp date 2020-04-30)
Anti-FLAG (M2, Sigma F3165 in PBS, use it directly without desalting) was recovered from the post-coupling antibody mix from 07/08/20. Original 750ug of anti-FLAG was used to make 75mg of anti-FLAG beads. After conjugation, antibody mix was saved, split into 3 tubes (~500ul each), put in speed vac for 1hr and the final volume was reduce to ½ (AmSO4 concentration increased from 1M to 2M). anti-FLAG precipitated out.
Take the tubes out from the speed vac and spin @ 14k rpm for 10’
Discard the supernatant, resuspend the pellet in 300ul of 0.1M NaPhosphate, pH7.4
Check the protein concentration by Bradford Assay, it was 1.02mg/ml
Use the recovered anti-FLAG and uncoupled beads from ERIBA to make more anti-FLAG beads
Antibody mix: 10ug of anti-FLAG antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads (make antibody mix for 75 mg; use 1/3 for 25mg of beads, 2/3 for 50mg reaction)
300 ul anti-FLAG antibody (1.02 mg/ml)
200 ul 3M AmSO4 (final concentration 1M)
100 ul 0.1M NaPO4, pH7.4
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, 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
Nup98-3xFLAG Anti-FLAG IP to test anti-FLAG beads
Date: 07/15/20
Cell lines: Nup98-3xFLAG (Dox induced, suspension 01/28/14)
Buffer: 20mM HEPES pH 7.4, 300mM NaCl, 1% Triton X-100
100mg scale with 10ul of either anti-FLAG beads
Resuspend in 0.5ml extraction buffer with protease inhibitors, 1:5 w/v
Vortex to mix
Sonicate 3 sec at setting 3
Spin @ 14k rpm, 4°C for 10’ (Eppendorf Centrifuge 5417R)
IP@ 4°C for 30’ (10ul slurry per 100mg powder)
Wash beads with 3x 1ml extraction buffer
Elute with 10ul 1x LDS @ 70°C for 5’
Add 50mM DTT to all samples
Heat samples @ 75°C for 10’
15-well 4-12% Bis-Tris gels, loading scheme as the following:
1) Marker
2) Nup98-3xFLAG_Old beads
3) Nup98-3xFLAG_Leila’s beads
4) Nup98-3xFLAG_New beads
5) BSA 50ng
6) BSA 150ng
