Conjugation of mouse and rabbit IgG Dynabeads
Date: 02/24/21
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
Make 100mg of beads with each antibody
Antibody mix: 10ug of antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads (for 100mg of beads, 1mg of antibody in 2000ul of antibody mix)
Mouse IgG
IgG from mouse serum (Sigma, V5381), reagent grade, ≥95% (SDS-PAGE), lyophilized powder. Lyophilized from 0.01 M phosphate buffer, pH 7.2, with 0.015 M NaCl
1mg per vial, dissolve in 200ul of in 0.1M Sodium Phosphate, pH7.4, enough to make 100mg of beads
200ul mIgG (5mg/ml)
667ul 3M AmSO4 (final concentration 1M)
1133ul 0.1M NaPO4, pH7.4
Rabbit IgG
Affinity Purified from Innovative Research IR-RB-GF-717. This is a frozen liquid buffered in 0.02M Sodium Phosphate; 0.15M NaCl; pH 7.4, @ 13.5mg/ml
75ul rabbit IgG (1000ug @ 13.5mg/ml)
667ul 3M AmSO4 (final concentration 1M)
1258ul 0.1M NaPO4, pH7.4
Filter the resulting antibody mix using Costar Spin X column (0.45um)
• Load 500ul on each column (500ul capacity; The column can be use repeatedly for the same antibody mix. Load the column multiple times if the volume is more than 500ul)
• Spin 12,000g, 1min (16,000g maximum speed)
Take 2ul of antibody mix before coupling
Split the beads pre-equilibrated in 0.1M NaPO4, pH7.4 into several aliquots; add antibody mix to the beads, mix well
25mg beads per tube (500ul of Ab mix per tube); 4 tubes
Leave the tube on a thermomixer at 37°C , O.N. (18 - 24hr)
Take 2ul of antibody mix after coupling
Wash the beads according to the Rout lab protocol Beads are resuspended in 50% Glycerol/1xPBS/0.5mg/ml BSA (667ul per 100mg of beads)
Make 100ul aliquots of beads when stored at -20°C
Testing new rIgG and mIgG beads
Date: 02/25/21
Cell line: LD401 (1x 50mg LD401Light; 6x 50mg of LD401 Puro 2.5, 9/3/2012, previously showed no ORF2 expression using anti-FLAG beads)
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale and powder to beads ratio: 50mg per IP reaction with 5ul of beads
Weigh out 50mg of LD401 Light powder and 2x 150mg of LD401 puro 2.5, add extraction buffer to each tube (1:4 w/v), vortex to mix
Sonicate 3 sec at setting 3
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Combine clarified lysate from two LD401 Puro2.5 tubes; Split supernatant evenly into 6 aliquots (50mg each)
Set up 7x 50mg IP reactions with 5ull of each kind of beads
1) LD401 Light_anti-ORF2
2) LD401 Puro_anti-ORF2
3) LD401 Puro_rIgG Old (10/04/19)
4) LD401 Puro_rIgG New
5) LD401 Puro_anti-ORF1
6) LD401 Puro_mIgG Old (10/22/19)
7) LD401 Puro_mIgG New
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute with 10ul LDS @ RT for 10’ with mixing
Elute again with 10ul LDS @70 °C for 5’ with mixing
Add DTT to 50mM to LDS elution and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze the elution
1) Marker
2) rIgG antibody mix_pre-coupling
3) rIgG antibody mix_post-coupling
4) mIgG antibody mix_pre-coupling
5) mgG antibody mix_post-coupling
6) Marker
7) LD401 Light_anti-ORF2
8) LD401 Puro_anti-ORF2
9) LD401 Puro_rIgG Old
10) LD401 Puro_rIgG New
11) LD401 Puro_anti-ORF1
12) LD401 Puro_mIgG Old
13) LD401 Puro_mIgG New
14) BSA_50ng
15) BSA_150ng
