Protein A Agarose IP Reagent

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Catalogue No: abx097210
Price: US$232.00
(Size: 2 ml)
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Protein A Agarose IP Reagent

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Datasheet SDS
Protein A Agarose IP Reagent is an agarose conjugate for use in immunoprecipitation. It is pre-blocked with BSA to reduce non-specific immunoglobulin binding.

Tested Applications IP
Recommended dilutions IP: 20 µl (resuspended volume)/test.
Form Liquid
Storage Store at 4°C. Do not freeze.
Buffer The product is provided as 0.5 ml agarose in 2.0 ml PBS.
Specificity Suitable for immunoprecipitation of mouse IgG2a, IgG2b and IgA; rabbit IgG; and human IgG1, IgG2 and IgG4.
Availability Shipped within 7-12 working days.
Note This product is for research use only.
Directions for use
  1. Incubate cultured cells (80-90% confluent monolayer in 100 mm cell culture plate or approximately 2-5 × 107 suspension cells in a flask) in methionine-free medium containing 5% dialysed fetal calf serum for 1 hour at 37 °C. The same procedure can be used for cells labeled with other radioactive amino acids (e.g. 14C or 3H) or with [gamma 32P]-orthophosphate. Cell labeling must be carried out in a medium lacking the relevant amino acid or in phosphate-free medium.
  2. Remove medium and replace with 3 ml methionine-free medium containing 5% dialysed fetal calf serum and 100 µCi/ml 35S-methionine. Incubate 1 hour at 37 °C. For some proteins, a longer labeling period (up to 18 hours) is preferable.
  3. Carefully remove radioactive medium with a pipette and wash the cell monolayer with PBS.
  4. Add 3 ml ice-cold RIPA buffer to the cell monolayer and incubate at 4 °C for 10 minutes. For suspension cells, add the RIPA buffer to the washed cell pellet in a 15 ml conical centrifuge tube.
  5. Disrupt cells by repeated aspiration through a 21 gauge needle and transfer to a 15 ml conical centrifuge tube.
  6. Wash the cell culture plate with 1 ml ice-cold RIPA buffer and combine with the original extract.
  7. Pellet cellular debris by centrifugation at 10,000 × g for 10 minutes at 4 °C. Transfer the supernatant to a new 15 ml conical centrifuge tube on ice. Preclear lysate (optional step) by adding 1 µg of the appropriate control IgG (normal mouse, rat, rabbit or goat IgG corresponding to the host species of the primary antibody) together with 20 µl of resuspended volume of Protein A&G-Agarose. Incubate at 4 °C for 30 minutes.
  8. Pellet beads by centrifugation at 2500 RPM (approximately 1000 × g) for 5 minutes at 4 °C. Transfer the supernatant (cell lysate) to a new 15 ml conical centrifuge tube on ice.
  9. Transfer 1 ml of the above cell lysate or approximately 100-500 µg total cellular protein to a 1.5 ml microcentrifuge tube. Add 1-10 µl (i.e. 0.2-2 µg) primary antibody (the optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4 °C.
  10. Add 20 µl of resuspended volume of Protein A&G-Agarose. Cap tubes and incubate at 4 °C on a rocker platform or rotating device for 1 hour to overnight.
  11. Collect immunoprecipitates by centrifugation at 2500 RPM (approximately 1000 × g) for 5 minutes at 4 °C. Carefully aspirate and discard the radioactive supernatant.
  12. Wash pellet 4 times with 1 ml RIPA buffer (more stringent) or PBS (less stringent) each time repeating centrifugation step above.
  13. After the final wash, aspirate and discard the supernatant and resuspend pellet in 40 µl of 1X electrophoresis sample buffer.
  14. Boil samples for 2-3 minutes and analyse 20 µl aliquots by SDS-PAGE and autoradiography. Unused samples may be stored at -20 °C.
  15. Optional: After boiling, samples may be centrifuged to pellet the agarose beads followed by SDS-PAGE analysis of the supernatant.
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