Conjugation of anti-ORF1, anti-GFP and anti-FLAG Dynabeads
Date: 08/16/21
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
Beads from NL (lot #00886548, expiration date 2021-11-30) and RU (lot #00938825, expiration date 2022-01-31)
Anti-ORF1 beads
Combine the rest of two batches of Abmart (5310-1-4M1234 4H1-140313, 8/11/19 400ul @2mg/ml and 11/19/19 1ml @ 1mg/vial)
Desalt into 0.1M NaPhosphate, pH7.4, using 3 Zeba 7k columns (0.5ml) follow the manufacturer’s instruction
Check the antibody concentration by Bradford Assay (1.1mg/ml; 1.4ml total)
1 tube of anti-ORF1 antibody recovered from old antibody mix; 1.76mg/ml, 340ul in 0.1M NaPhosphate
Total anti-ORF1 antibody: 1.4ml @ 1.1mg/ml and 0.34ml @ 1.76mg/ml (2.1mg, enough to make 140mg of beads)
Beads used: 100mg from NL and 40mg from RU (equilibrated with 0.1M NaPhosphate)
Antibody mix: 20ul per mg of beads, 1ml total vol. use 15ug of anti-ORF1 antibody per mg of dynabeads; total volume of antibody mix 2800ul
1740ul anti-ORF1 antibody (15ug Ab / mg beads, 2.1mg anti-ORF1 total)
933ul 3M AmSO4 (final concentration 1M)
127ul 0.1M NaPO4, pH7.4
Anti-GFP beads
Llama anti-GFP antibody from Sam, 1mg/ml in 1x PBS, pH7.4, can be directly used for conjugation; use 1mg of antibody to make 100mg of beads (beads are from NL)
1000ul anti-GFP antibody (10ug Ab / mg beads,1mg antibody)
667ul 3M AmSO4 (final concentration 1M)
333ul 0.1M NaPO4, pH7.4
The antibody mix looked cloudy. I span it down and saw white pellet on bottom of the tube. According to Sam, antibody precipitated out is not unusual. He normally uses the cloudy antibody mix directly. During conjugation, antibody will couple to the beads which reduces antibody concentration in the mix. At the end of the conjugation, antibody mix would become clear.
After 18hrs, take half of the anti-GFP beads (50mg, two tubes with 500ul of antibody mix) and start wash steps. Label this half of beads as “anti-GFP 1x”, one overnight coupling. Put the other two tubes (2x 25mg conjugation reaction) on the magnet, take the supernatant; dissolve the antibody pellet with the supernatant and Resuspend the antibody pellet (from the day before) with antibody mix and add antibody mix back to the beads. Let the conjugation continue O.N. again. Label the beads as “anti-GFP 2x” (two overnight coupling)
Anti-FLAG beads
Anti-FLAG (M2, Sigma F3165 in PBS, 4mg/ml); take 2mg to make 200mg of beads (100mg from NL and 100mg from RU)
500ul anti-FALG antibody (10ug Ab / mg beads,2mg antibody)
1333ul 3M AmSO4 (final concentration 1M)
2167ul 0.1M NaPO4, pH7.4
All beads are equilibrating with 0.1M NaPO4, pH7.4
Make 20 or 25mg aliquots based on amounts of beads to make
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 hr)
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
8/17/21 More beads
Anti-GFP beads
Got another tube of Llama GFP antibody from Paula, also @ 1mg/ml. Make another 25mg of beads; beads are from RU)
250ul anti-GFP antibody (10ug Ab / mg beads,1mg antibody)
167ul 3M AmSO4 (final concentration 1M)
83ul 0.1M NaPO4, pH7.4
This time, the antibody mix was clear.
Conjugation was done @ 30C for 18h this time (this is the temperature we used in the past. Sam and others in the Rout lab also use 30C to make anti-GFP beads)
Anti-ORF2 beads (also see
Anti-ORF2 antibody (clone 9-7) from Abmart, 1mg/ml in 1x PBS, pH7.4, can be directly used for conjugation; use 0.5mg of antibody to make 50mg of beads (beads are from RU)
500ul anti-GFP 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
Gel 1: Ab mix of anti-ORF1, anti-GFP and anti-FLAG
From left to right (forgot load marker on this gel):
• Anti-ORF1_before coupling
• Anti-ORF1_after (NL)
• Anti-ORF1_ after (RU)
• Anti-GFP_before coupling
• Anti-GFP_after (NL)
• Anti-FLAG_before coupling
• Anti-FLAG_after (NL)
• Anti-FLAG_ after (RU)
Gel 2: Ab mix of anti-ORF2 and anti-GFP (coupling @30C), sypro stain
Loading:
1) BSA_50ng
2) BSA_100ng
3) Space
4) Unstained marker_1ul
5) Anti-ORF2_before coupling
6) Anti-ORF2_after coupling
7) Space
8) Anti-GFP_before coupling
9) Anti-GFP_after coupling (NL)
10) Prestained marker_5ul
Testing anti-ORF1, anti-GFP and anti-FLAG beads
Date: 08/18/21
Cell lines:
N2102Ep to test anti-ORF1 beads; 25mg scale with 5ul of beads (3x 25mg reactions)
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl
Anti-ORF1_old (5/27/21)
Anti-ORF1_new_NL (8/17/21)
Anti-ORF1_new_RU (8/17/21)
CBP80-LAP (from Yuhui 03/24/16) to test anti-GFP beads; 50mg with 5ul of beads (4x 50mg reactions)
Extraction/wash buffer: 20mM HEPES, pH7.4, 0.5% Tritonx-100, 150mM NaCl
Anti-GFP_old (3/8/21)
Anti-GFP_1x (8/17/21)
Anti-GFP_2x (8/18/21)
Anti-GFP_30C (8/18/21)
Z18-3x FLAG (4/19/16) to test anti-FLAG beads; 50mg with 5ul of beads (3x 50mg reactions)
Extraction/wash buffer: 20mM HEPES, pH7.4, 0.5% Tritonx-100, 600mM NaCl
Anti-FLAG_old (10/16/20)
Anti-FLAG_new_NL (8/17/21)
Anti-FLAG_new_RU (8/17/21)
Weigh out powder as needed, add appropriate extraction buffer with protease inhibitors (1:4 w/v)
Vortex to mix
Sonicate 2 sec x 5 @ 2 Amp (total energy output was 21J)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Transfer supernatant to fresh tubes, combine 4x 250mg preps into one tube
Incubate @ 4°C for 30 on rotating wheel
After IP step, wash beads with 3x 3ml extraction buffer (transfer the beads to fresh tubes during 2nd wash)
Elute with 10ul of 1.1x LDS (70°C for 5’ with mixing)
Add DTT to each sample (50mM final concentration)
Heat samples @ 75°C for 10’
Load elution on 15-well 4-12% Bis-Tris gel
1) Marker
2) Anti-ORF1_old
3) Anti-ORF1_new_NL
4) Anti-ORF1_new_RU
5) Anti-FLAG_old
6) Anti-FLAG_new_NL
7) Anti-FLAG_new_RU
8) Anti-GFP_old
9) Anti-GFP_1x
10) Anti-GFP_2x
11) Anti-GFP_30C
12) BSA_50ng
13) BSA_150ng
New anti-FLAG beads (both NL beads and RU beads) are fine. New anti-ORF1 beads have high antibody leakage. New anti-GFP beads also have higher antibody leakage comparing to old beads, but IP yield are higher than old beads as well.
Take one aliquot (100ul) of the following beads, repeat all washing step of the conjugation protocol or 100mM Glycine, pH3.5 wash (protocol for regenerate beads after native elution)
Regenerate beads after native elution (for each 100ul beads):
Quick wash with 0.5ml 100mM Glycine, pH3.5
(Vortex to mix, brief spin, put the tube on magnet and take off the Glycine)
Wash beads with 0.5ml 100mM Glycine, pH3.5 @ RT for 2 min
(Add glycine, vortex to mix and leave the tube on a mixer for 2min)
Wash beads with 0.5ml of 1x PBS with 0.5% Triton X-100
Quick wash with 0.5ml 100mM Glycine, pH3.5
Wash beads with 0.5ml 100mM Glycine, pH3.5 @ RT for 2 min
Wash beads 3 time with 1ml of 1x PBS with 0.5% Triton X-100
Wash beads once with 1ml of 1x PBS
Discard 1x PBS
Add 80ul of 1xPBS + 50% glycerol + 0.5mg/ml BSA, mix with beads; Store beads @ -20°C
1) Anti-ORF1_NL
2) Anti-GFP_2x
3) Anti-GFP_2x (glycine wash)
After wash, add storage buffer back to beads. Take 5ul of beads with extra wash and 5ul of same beads without extra beads, wash 3x 0.5ml 1xPBS and elute with 10ul 1.1x LDS first @ RT for 10’; collect the eluate and repeat elution with 1.1x LDS first @70C for 5’; collect eluate again; Run RT elution and 70C elution side by side
1) Marker
2) E_Anti-ORF1_NL_no extra wash_RT
3) E_Anti-ORF1_NL_with extra wash_RT
4) E_Anti-GFP_2x_no extra wash_RT
5) E_Anti-GFP_2x_with extra wash_RT
6) E_Anti-GFP_2x_with glycine wash_RT
7) E_Anti-ORF1_NL_no extra wash_70C
8) E_Anti-ORF1_NL_with extra wash_70C
9) E_Anti-GFP_2x_no extra wash_70C
10) E_Anti-GFP_2x_with extra wash_70C
11) E_Anti-GFP_2x_with glycine wash_70C
12) BSA_50ng
13) BSA_100ng
Anti-ORF1 IP testing beads with extra wash
N2102Ep to test anti-ORF1 beads; 25mg scale with 5ul of beads (2x 25mg reactions)
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl
Anti-ORF1_old (5/27/21)
Anti-ORF1_new_NL (8/17/21) with extra wash
Weigh out 50mg of N2102Ep powder, add appropriate extraction buffer with protease inhibitors (1:4 w/v)
Vortex to mix
Sonicate 4 sec x 1 @ 2 Amp (total energy output was 11J)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Transfer supernatant to fresh tubes, combine 4x 250mg preps into one tube
Incubate @ 4°C for 30 on rotating wheel
After IP step, wash beads with 3x 3ml extraction buffer (transfer the beads to fresh tubes during 2nd wash)
Elute with 10ul of 1.1x LDS (RT for 10’ with mixing)
Collect eluate (E_RT)
Elute again with 10ul of 1.1x LDS (70°C for 5’ with mixing)
Collect eluate (E_70C)
Add DTT to each sample (50mM final concentration)
Heat samples @ 75°C for 10’
Load elution on 15-well 4-12% Bis-Tris gel
1) Maker
2) E_RT_Old anti-ORF1 beads (5/27/21)
3) E_RT_New anti-ORF1 beads (8/17/21 with extra wash)
4) E_70C_Old anti-ORF1 beads (5/27/21)
5) E_70C_New anti-ORF1 beads (8/17/21 with extra wash)
When we checked beads with extra wash by just eluting beads with LDS @ RT and 70C without IP, we know that extra wash can reduce antibody leakage but did not prevent the leakage completely. Antibody leakage is not a big problem when elution was done @ RT. But ORF1p and ORF2p can only be fully eluted from beads @ 70C. This new batch of beads is not as good as previous batch. Note: previous batch of anti-ORF1 beads (5/27/21) was made with anti-ORF1 recovered from old antibody mix.