I am doing a coIP, but I am stuck on a step with the protocol. The protocol is as follows:
Washing & Usage of Gamma binding beads
- In a falcon tube, take 15 ml 1x (FS) PBS.
- Take the Gamma-bind Sepharose beads from the fridge, shake it gently.
- Cut a 1 ml pipette tip at the edge and pipette 100 µl of beads (for 2 tubes) from the stock and place it at the bottom of the PBS falcon tube. You need to adjust the number of beads to be taken, according to your experiment.
- Spin the tube containing the beads in PBS at 2000g for 2 min.
- Use a serological pipette to carefully remove the PBS until you reach 1 ml. Once you reach the 1 ml mark, use a fine tip (tips used for loading SDS-PAGE gels), insert the tip into the beads and remove the PBS until it's dry.
- Add 1 ml of IP buffer to the beads, spin down the tubes at 2,000 g for 2 min and rotate it for 5 min. (Incubate on ice, when not on shaker).
In the last step (#6), should I discard the supernatant as the next step is to add the beads to antibody-lysate complex?
Should I try to scoop the beads from the bottom of the tube or should I take ~ 40-50 ul of the beads with the IP buffer and add it to my ab-lysate? If I use 40-50 ul, the rest of the beads with IP buffer can be discarded or can I store it at 4 degrees and use it for other experiments?