Conjugation of anti-PRCAKA to Dynabeads
Materials: Beads: thermo M270 Epoxy Antibody: Cell signallingTech., PKA C-alpha antibody (038C6) Rabbit mAb,1.3mg/ml
Regents:
0.1 M NaPO4 buffer, pH7.4 3 M Ammonium sulfate 100 mM Glycine HCl, pH2.5 10 mM Tris, pH8.8 100 mM Triethylamine (Make fresh by adding 168ul stock to 11.156mL of DDH20) PBS with 0.5% Triton X-100
Protocol:
Day1:
Equilibrate antibody with 0.1 M NaPO4 using 7kDa Zeba Spin columns, the protocol is followed the user guide. (Skip this step since there is no need for desalting for this antibody)
Measure protein concentration using 660 nm assay. >> 2 mg/ml Total 200 ug/100uL of antibody , (used 5ul for 660nm assay)
Weight out appropriate amount of Dynabeads (10 ug antibody/mg beads) Weight out 20mg of beads
Equilibrate beads with 1.06 ml of 0.1 M NaPO4 (1.6 mL of buffer/ 30 mg beads) vortex to mix, shake slowly on mutator or end to end mixer for 10 min While beads suspension is on Nutator, Prepare AB MIX. (20 uL of reaction buffer /mg beads)
Total 400uL of AB MIX is needed. And the final concentration of AmSO4 is 1M. 100 uL of antibody 167 uL of 0.1 M NaPO4 133.33 uL of 3M AmSO4, drop by drop with eppendorf pipette. Last one to add, avoid precipitation. save 2 uL of antibody mix before coupling for gel analysis
Place the step5 tube with the bead suspension onto magnetic holder and wait until all beads are attached to the magnet. Bead solution will appear clear. Aspirate the buffer off (be careful not to aspirate off the beads too).
Add AB mix to beads pre-equilibrated with 0.1M naPO4, PH7.4; vortex to completely combine the AB mix and the beads
Place Thermomixer in 37C incubator overnight (18-24 hours) for 22 hours
Day2
save the supernatant after coupling, use 2uL for gel analysis (Check the depletion of antibody during coupling by running antibody mix before and after coupling on gel (2ul each)), keep the rest in 4C , recover antibody from FT using Speedvac
Wash Wash beads 1X , 1mL of 100mM Glycine HCl pH2.5. Put it on and take it off as fast as possible
Wash 1X with 1mL of 10mM Tris pH 8.8. Wash 1X with 1mL of fresh 100mM Triethylamine. Put it on and take it off as fast as possible.( 168ul TEA to 11.156ml H2O under hood).
Wash 4X with 1mL of PBS for 5’
Wash 1X with PBS + 0.5% Triton X-100 for 5’
Wash 1X with PBS +0.5% Triton X-100 for 15 minutes on rocker/nutator.
Resuspend all beads in 50% Glycerol/1xPBS/0.5mg/ml BSA (final Conc: 150 mg of beads/mL), save at -20C