Protein G Agarose IP Reagent
REQUEST MORE INFO
Price:
US$232.00
(Size: 2 ml)
Click on the image to see the image legend
Protein G 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 IgG1, IgG2a, IgG2b and IgG3; rat IgG1, IgG2a, IgG2b and IgG2c; rabbit and goat polyclonal antibodies; and human IgG1, IgG2, IgG3 and IgG4. |
Availability |
Shipped within 7-12 working days. |
Note |
This product is for research use only. |
Directions for use |
- 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.
- 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.
- Carefully remove radioactive medium with a pipette and wash the cell monolayer with PBS.
- 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.
- Disrupt cells by repeated aspiration through a 21 gauge needle and transfer to a 15 ml conical centrifuge tube.
- Wash the cell culture plate with 1 ml ice-cold RIPA buffer and combine with the original extract.
- 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.
- 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.
- 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.
- 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.
- Collect immunoprecipitates by centrifugation at 2500 RPM (approximately 1000 × g) for 5 minutes at 4 °C. Carefully aspirate and discard the radioactive supernatant.
- Wash pellet 4 times with 1 ml RIPA buffer (more stringent) or PBS (less stringent) each time repeating centrifugation step above.
- After the final wash, aspirate and discard the supernatant and resuspend pellet in 40 µl of 1X electrophoresis sample buffer.
- Boil samples for 2-3 minutes and analyse 20 µl aliquots by SDS-PAGE and autoradiography. Unused samples may be stored at -20 °C.
- Optional: After boiling, samples may be centrifuged to pellet the agarose beads followed by SDS-PAGE analysis of the supernatant.
|
Research Articles on Protein G Agarose IP Reagent