CONJUGATION OF DYNABEADS Dynabeads M270 Epoxy (Invitrogen) (Modified from Rout Lab protocol: http://lab.rockefeller.edu/rout/assets/file/protocols/Conjugation_of_Dynabeads.pdf)
Date: 23/09/19
Follow the Rout lab Dynabeads conjugation protocol linked above (make necessary modifications based on the amount of beads and antibody used)
Antibody: Sigma F3165: Anti-FLAG Clone M2 mouse monoclonal antibody Concentration: ~4mg/ml (4ug/ul) Note: We use directly without de-salting. This product is more expensive than the alternative Sigma F1804. However, F1804 requires double desalting. After column loss (and column costs) we determined that the cost savings is not significant and clone M2 performs as well or better. (see Hua’s experiment INSERT WIKI LINK)
Day One – Preparing the Beads and Conjugation
1. Weigh out the appropriate amount of Dynabeads, Equilibrate to 0.1M Na Phosphate buffer pH 7.4 (16mL of buffer per 300mg beads). 100mg beads = 5.3ml buffer 2. Divide bead suspension into aliquots in appropriate number of tubes 3. Vortex 30 seconds to mix 4. Shake slowly for 10 minutes on Nutator or end-over end rotator 5. While bead suspension is incubating prepare the antibody mix (AB mix) 6. Set up the CONJUGATION reaction in the following order (this will be the AB mix):
For 100mg of beads, the ideal reaction volume is 2ml (20ul per mg of beads). Mix will be prepared in a 14mL culture tube to facilitate addition of ammonium sulfate.
Take 250ul of antibody (1mg) to make 100mg of beads, 666ul of 3M AmSO4 (final concentration 1M), and 1084ul of 0.1M NaPhosphate, pH7.4
in a 14mL culture tube, add 3M AmSO4 one drop at a time to the tube while mixing at very low speed on vortex
X ul Antibody (10ug antibody / mg beads)
666 ul 3M (NH4)2SO4 (final concentration 1M)
1334-X ul 0.1M NaPhosphate, pH7.4
Save 1ul of antibody mix before coupling for gel analysis
7. Place the tubes with the bead suspension onto appropriate size 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). 8. Wash again with same volume of 0.1M NaPO4 - incubation for 10 minutes is not necessary. Vortex 15 seconds. Put on the Magnet - aspirate off the buffer. 9. Add AB mix to low-bind safe-lock Eppendorf tube; vortex to completely combine the AB mix and the beads 10. Place in 37C Thermomixer with 1000rpm shaking 11. Place Thermomixer in 30 or 37C incubator overnight (18-24 hours)
Day Two –Washing Dynabeads after Conjugation
Save entire supernatant (FT) after coupling, use 1ul for gel analysis
Wash the beads according to the Rout lab protocol:
Washes should be aspirated using a Vacuum Aspirator for maximum efficiency 1) Wash once with 1mL of 100mM Glycine HCL pH2.5. Put it on and take it off as fast as possible. 2) Wash once with 1mL of 10mM Tris pH 8.8. 3) Wash once with 1mL of 100mM Triethylamine. (Make fresh 100 mM Triethylamine by adding 168ul stock to 11.156mL of DDH20). Put it on and take it off as fast as possible. 4) Wash the coated beads with 1x PBS for 5 minutes – washes should be done on a rocker/nutator – repeat 4x. (FIVE total washes this step) 5) Wash once with PBS + 0.5% Triton X-100 for 5 minutes. 6) Wash again with PBS +0.5% Triton X-100 for 15 minutes on rocker/nutator.
Beads are resuspended in the following buffer for long-term storage at -20°C 50% Glycerol/1xPBS/0.5mg/ml BSA
Make 100ul aliquots of beads when stored at -20°C
Check the depletion of antibody during coupling by running antibody mix before and after coupling on gel (1ul each)
4-12% Bis-Tris gel, loading order
1) Marker
2) F3165_AbMix_PreCoupling
5) F3165_AbMix_PostCoupling
6) BgG_50ng
7) BgG_150ng
8) Marker
Necessary Solutions 0.1M Sodium Phosphate Buffer (NaPO4) – pH 7.4 2.62g NaH2PO4 x H2O (MW 137.99) 14.42g Na2HPO4 x 2H2O (MW 177.99) Dissolve in distilled water, adjust pH if necessary and adjust to 1 liter. This buffer is for prewashing beads, do not add any protein, sugar, etc.
3M Ammonium Sulfate (stock solution) 39.6g (NH4)2SO4 (MW 132.1) Dissolve in 0.1M Sodium Phosphate Buffer (pH 7.4) and adjust to 100mL
Phosphate Buffered Saline (PBS) - pH 7.4 0.26g NaH2PO4 x H2O (MW 137.99) 1.44g Na2HPO4 x 2H2O (MW 177.99) 8.78g NaCl (MW 58.5) Dissolve in 900mL distilled water, adjust pH if necessary and adjust to 1 liter.
PBS + 0.5% Triton X-100 Include 0.5% (w/v) Triton X-100 in 100 mL PBS solution
100mM Glycine HCl pH 2.5 10mM Tris pH 8.8 10% Sodium Azide (NaN3)