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SY SY - Synaptic Systems SY SY - Synaptic Systems

Protocol for immunoprecipitation

Solutions needed:

Buffer A

  • 10 mM Tris/HCL (pH 7.4)

  • 150 mM NaCl

  • 1 % Triton-X100

  • Protease inhibitors


Buffer B

  • 10 mM Tris/HCL (pH 7.4)

  • 150 mM NaCl

  • 0.2 % Triton X100

  • Protease inhibitors


Buffer C

  • 10 mM Tris/HCL (pH 7.4)

  • Protease inhibitors

Procedure:

  1. Solubilize proteins by incubation in buffer A if necessary.
    Starting material should have a concentration of 1-4 mg/ml.

  2. Bind 5-10 µg of antibody to 25 µl Protein-G or A slurry by incubation in 200 µl buffer C for 2 h.

  3. Block beads for 30 min with 2 % BSA in buffer C for 30 min.

  4. Wash beads with buffer C.

  5. Add 100-200 µl sample and incubate for 2-4 h at 4°C rotating head over tail.

  6. Wash suspension twice with buffer B.

  7. Wash suspension with buffer C.

  8. Incubate pellet with SDS loading buffer and apply to SDS PAGE.

Some proteins are not efficiently solubilized by NP40 or triton. For these proteins the solubilization protocol described in Geumann et al. should be employed:

Geumann C, Grønborg M, Hellwig M, Martens H & Jahn R (2010). A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins. Analytical Biochemistry 402: 161-9.


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Synaptic Systems GmbH | Rudolf-Wissell-Str. 28 | 37079 Göttingen | Germany
Phone: +49 (0)551/505 56-0 | Internet: http://www.sysy.com | E-Mail: sales@sysy.com