LaCava Research Wiki

Test new Dynabeads

Dynabeads M270 Epoxy (Invitrogen)

Antibodies:
Marty Taylor anti-FLAG M2, Sabatini Lab Concentration: ~17mg/ml
Sigma F3165: Anti-FLAG Clone M2 mouse monoclonal antibody Concentration: ~3.5mg/ml
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 F3165 performs as well or better. Both antibodies are derived from clone M2. (see Hua’s experiment INSERT WIKI LINK)

Day One – Preparing the Beads and Conjugation
Perform these steps in the afternoon and wash the conjugated beads the next morning.

1. Remove beads from +4C, allow to equilibrate to RT
2. Weigh out 50mg of Dynabeads, Equilibrate to 0.1M
3. 50mg will be divided in half, 25mg to be conjugated with Marty’s anti-FLAG, the other 25mg to be conjugated with SIGMA F3165 anti-FLAG
4. Na Phosphate buffer pH 7.4 (16mL of buffer per 300mg beads). 100mg beads = 5.3ml buffer (50mg beads = 2.65ml; 25mg beads = 1.325ml)
5. Weigh 50mg aliquot of unconjugated beads into a 2ml locking eppendorf tube then add 1.32ml of NaPO4
6. Vortex 30 seconds to mix
7. Shake slowly for 10 minutes on Nutator or end-over-end rotator
8. While bead suspension is incubating prepare the antibody mix (AB mix)
9. 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 have 2x25mg; each tube of 25mg will get 500ul Ab mix. Prepare 500ul of each type of Ab mix (Marty and Sigma) then divide in half between ERIBA and RU beads.

SIGMA Ab Mix ~286ul of antibody (1mg) per 100mg of beads; so we will use 71.5ul of antibody for 25mg of beads. First combine the antibody and the NaPO4, then AmSO4 is added dropwise while vortexing. 167ul of 3M AmSO4 (final concentration 1M), and 261.5ul of 0.1M NaPhosphate, pH7.4

Marty Ab Mix ~59ul of antibody (1mg) per 100mg of beads; so we will use 14.7ul of antibody for 25mg of beads. First combine the antibody and the NaPO4, then AmSO4 is added dropwise while vortexing.

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).

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.

Add AB mix to low-bind safe-lock Eppendorf tube; vortex to completely combine the AB mix and the beads

Place in end-over-end rotator

Place rotator in 37C incubator overnight (18-24 hours)

Day Two –Washing Dynabeads after Conjugation
Save entire supernatant (FT)at -20C after coupling

Before beginning prepare fresh triethylamine (Make fresh 100 mM Triethylamine by adding 168ul stock to 11.156mL of DDH20)

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. 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:

• 5ml of 100% glycerol
• 1ml of 10x PBS
• 2.5ml of 2mg/ml BSA
• Add H2O up to 10ml total vol
• Mix well
• Filter the resulting buffer and store at -20°C

Make 100ul aliquots of beads when stored at -20°C

1/10/20 beads testing gel

Cell lines: SMAD-3xFlag (~65 kD), RRP6-3xFlag (new) (~110)

Controls:
-Eriba beads
-Hua beads

Buffer: 300 mM NaCl 20mM HEPES 1% Triton

IP for 30 min of 4C rotating: New Marty, Old Marty, New Sigma, Old Sigma, Hua

Mock elution: Beads were washed 3x with 300 mM NaCl 20mM HEPES 1% Triton

Elute every IP (and mock too) with 10 ul 1.1 LDS 5 min 70C
Load on a gel with 50 ng and 150 ng of BSA

Loading order:

1. -
2. Marker
3. Hua RRP6
4. Hua SMAD
5. Hua Mock
6. Marty old RRP6
7. Marty old SMAD
8. Marty old Mock
9. Sigma old RRP6
10. Sigma old SMAD
11. Sigma old Mock
12. Marty new RRP6
13. Marty new SMAD
14. Marty new Mock
15. Sigma new RRP6
16. Sigma new SMAD
17. Sigma new Mock
18. BSA 50 ng
19. BSA 150 ng