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Preparation of Ni NTA beads tweezer0

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					                                                                  Kellermayer group. Ni-NTA beads




                              Preparation of Ni-NTA beads


Date:


Bead type:


BUFFERS:
1. 100 mM MOPS-KOH, pH 7.5
2. 10 mM NiCl2, 10 mM Glycine
3. 10 mM sulfo-EGS (Pierce) in MOPS-KOH (6.6 mg/1 ml buffer)
4. 100 mM AB-NTA (Dojindo) in MOPS-KOH (32.4 mg/1 ml buffer)
5. Assay buffer, i.e., the solution that you will use with your proteins or nucleic acids in the
tweezer


LABELING STEPS:
1. Centrifuge 0.244 ml amino-latex beads (2.1 µm, 4.1%) at 5000 rpm, 5 min (Eppendorf
tabletop fuge)
2. Resuspend pellet in 1 ml sulfo-EGS solution. This will give a bead concentration of 1 %.
3. Incubate (on Rotorack) beads at room temperature for 15 min
4. Centrifuge beads (5000 rpm, 5 min)
5. Resuspend pellet in 1 ml AB-NTA
6. Incubate (on Nutator) at room temperature for 60 min
7. Centrifuge beads
8. Resuspend pellet in 1 ml MOPS-KOH
9. Repeat steps 7-8 three times
10. Wash beads four times with NiCl2 solution (centrifuge-resuspend in 1 ml solution each
time)
11. Wash beads four times with MOPS-KOH
12. Wash beads four times with Assay buffer.
13. Dilute beads for laser tweezer experiment with Assay buffer.

				
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Description: Preparation of Ni NTA beads tweezer0