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Membrane Preparation Protocol

Note: The following protocol has been developed for use with GPCR Polyclonal Antibodies manufactured by Proteimax. Other antibodies have not been tested using this protocol. For additional assistance, please contact Technical Support.

Buffers to Prepare

Buffer A (must be prepared fresh for each use)
50 mM Tris-Cl, pH 7.4 + 1 mM EDTA + 10% sucrose

Buffer B
50 mM Tris-Cl, pH 7.4 + 1 mM EDTA

Buffer C
50 mM Tris-Cl, pH 7.4

Procedure

All operations should be carried out on ice.

  1. Dissect brain regions or spinal cord, estimate approximate weight and freeze immediately.
  2. Suspend the tissue in 40-fold ice-cold Buffer A.
  3. Homogenize the tissue by hand using a Dounce homogenizer until a homogeneous suspension with no particulate matter is achieved.
  4. Centrifuge for 10 minutes at 35,000 x g at 4°C. Discard the supernatant.
  5. Resuspend the pellet in 40 mL of Buffer B; ensure that the suspension is homogenous.
  6. Leave on ice for 30 min.
  7. Centrifuge for 20 min at 35,000 x g at 4°C.
  8. Resuspend the pellet in a minimum volume (~300 μL/10 spinal cords) of Buffer C. Pass through a fine (26 g) needle to ensure homogeneous suspension.
  9. Estimate protein concentration of the supernatant using a BCA assay.
  10. Store membranes at -70°C at a concentration of 5 μg/μL in Buffer C.
 

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