Coupling of TMEM192 antibody to MyOne™ Epoxy Dynabeads™
Dario R Alessi, Daniel Saarela, Pawel Lis
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Abstract
This protocol describes coupling of 600µg of rabbit monoclonal TMEM192 antibody (Abcam recombinant Anti-TMEM192 antibody [EPR14330-67], BSA and Azide free, ab232600) to 20mg of MyOne™ Epoxy Dynabeads™ (Invitrogen™, 34001D) to obtain 2mL of final suspension. The coupled beads generated using this protocol can be used for the isolation of untagged lysosomes from cells and tissues.
Attachments
Steps
Protocol
Before opening the vial containing dried magnetic beads, equilibrate to Room temperature.
Thaw the antibody On ice and keep On ice until it is needed in step 8.
Calculate the volume of antibody needed, so that 600µg is used – this volume should
be ≤500µL.
Weigh 20mg beads directly into a fresh low-binding 1.5 ml Eppendorf tube.
Resuspend beads in 1000µL of sterile Milli-Q water, vortex for 0h 0m 15s, sonicate in a water bath sonicator for 0h 5m 0s.
Place vial on a magnetic rack for 0h 1m 0s, remove water using a pipette.
Repeat steps 5 and 6. After sonication there should be no bead aggregates visible.
Add the required volume of antibody to the vial containing washed beads.
Add buffer C1 up to total volume of 500µL (C1= 500 - antibody volume). Vortex to resuspend the beads.
Add 500µL of buffer C2 and vortex.
Incubate in a Thermomixer at 37°C for 16–24 hours (typically 20h 0m 0s) at 1500rpm (make sure the beads do not settle).
Place on a magnetic rack for 0h 1m 0s, remove liquid using a pipette.
Resuspend beads in 1000µL of buffer HB, vortex.
Place on a magnetic rack for 0h 1m 0s, remove liquid using a pipette.
Resuspend beads in 1000µL of buffer LB, vortex.
Place on a magnetic rack for 0h 1m 0s, remove liquid using a pipette.
Resuspend beads in 1000µL of buffer SB, vortex.
Repeat steps 16 and 17:
- Place on a magnetic rack for
0h 1m 0s, remove liquid using a pipette. - Resuspend beads in
1000µLof buffer SB, vortex.
Place on a magnetic rack for 0h 1m 0s, remove liquid using a pipette.
Resuspend beads in 1000µL of buffer SB, vortex.
Incubate in a shaker at Room temperature for 1500rpm
Place on a magnetic rack for 0h 1m 0s, remove liquid using a pipette.
Resuspend beads in 1000µL of buffer SBS. At this stage beads are at 20mg/ml and should be stored in the fridge. Beads can be further diluted with buffer SBS to 10mg/ml, which is the usual working concentration.
