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CONJUGATION OF DYNABEADS Dynabeads M270 Epoxy (Invitrogen) (Modified from Rout Lab protocol: http://lab.rockefeller.edu/rout/assets/file/protocols/Conjugation_of_Dynabeads.pdf)

Follow the Rout lab Dynabeads conjugation protocol linked above (with modification based on the amount of beads and antibody used)

Antibody: Anti-GFP antibody (Green Lobster nanobody from Rout Lab; LaG16-G2 dimer) Concentration: 4.5mg/ml – perform Bradford Assay to confirm.

Antibody Mix for nanobody beads (30mg of beads for each nanobody) 15ul per mg of beads; 600ug of each nanobody; 1200ul total vol.

Nanobodies are in 20mM HEPEs, 150mM NaCl, pH7.4

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). 60mg beads = 3.2mL buffer
2. Divide bead suspension into aliquots in appropriate number of falcon/Eppendorf 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). We are making 60mg of beads, so ideal reaction volume is 1.2ml. Mix will be prepared in a 14mL culture tube to facilitate addition of ammonium sulfate.

Take desired amount of antibody, mix with calculated amount 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 low speed on vortex

X ul Nanobody (10ug antibody / mg beads)

400 ul 3M (NH4)2SO4 (final concentration 1M)

800-X ul 0.1M NaPhosphate, pH7.4

Save 1ul of antibody mix before coupling for gel analysis

1. 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).
2. 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.
3. Add AB mix to low-bind safe-lock Eppendorf tube; vortex to completely combine the AB mix and the beads
4. Place in 37C Thermomixer with 1000rpm shaking
5. Place Thermomixer in 30 or 37C incubator overnight (18-24 hours)

Save the supernatant after coupling, use 1ul for gel analysis

Wash the beads according to the Rout lab protocol

Beads are resuspended in 50% Glycerol/PBS, pH7.4/ 0.02% NaN3

Make 100ul aliquot and keep at -20°C for long-term storage

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. LaG16-G2-10_AbMix_PreCoupling
3. LaG16-G2_AbMix_PostCoupling
4. BSA_50ng
5. BSA_150ng
6. 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)