|Place of Origin||USA|
|Technical Information||View technical data|
Currently there is no direct assay to measure the activation of Arl1 GTPases.
NewEast Biosciences Arl1 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Arl1-GTP, but not Arl1-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a short time. This assay provides the reliable results with consistent reproducibility.
NewEast Biosciences Arl1 Activation Assay Kit bases on the configuration-specific anti-Arl1-GTP monoclonal antibody to measure the active Arl1-GTP levels, either from cell extracts or from in vitro GTPγS loading Arl1 activation assays. Briefly, anti-active Arl1 mouse monoclonal antibody will be incubated with cell lysates containing Arl1-GTP. The bound active Arl1 will then be pulled down by protein A/G agarose. The precipitated active Arl1 will be detected by immunoblot analysis using anti Arl1 rabbit polyclonal antibody or anti-Arl1 mouse monoclonal antibody.
This antibody specifically recognizes Arl1-GTP from all vertebrates.
GDP-labeling of 0.5 mL of cell lysate.
Store all kit components at 4ºC until their expiration dates.
Materials Needed but Not Supplied
9. Collect the supernatant and store samples (~1-2 mg of total proteins) on ice for mediate use, orsnap freeze and store at - 70 °C for future use.
1. Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 µg of purified Arl1
2. To each tube, add 20 µl of 0.5 M EDTA (to 20 mM final concentration).
6. Stop loading by placing the tubes on ice and adding 32.5 µl of 1 M MgCl2 (to 60 mM,
8. Aspirate and discard the supernatant, making sure not to disturb/remove the bead
9. Wash the bead 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and
aspirating each time.
13. Centrifuge each sample for 10 seconds at 5,000 x g.
1. Load 15 µL/well of pull-down supernatant to a polyacrylamide gel (17%). Also,it’s recommended to include a pre-stained MW standard (as an indicator of a successful transfer instep 3).
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following the manufacturer’s instructions.
6. Use the detection method of your choice such as ECL.
Example of Results
Arl1 Activation Assay. Purified GST-tagged Arl1 proteins were immunoprecipitated with theanti-active Arl1 monoclonal antibody (Cat. # 26924) after treated with GDP (lane 1) or GTPγS (lane 2),and was blotted with anti-Arl1 monoclonal antibody (Cat. # 26056). Arl1 protein input control is shown in the bottom panel.
|26924||Active Arl1-GTP Monoclonal Antibody||30 μL||$495|
|82901||Arl1 Activation Assay Kit||20 assays||$737|
|26056||Anti-Arl1 Mouse Monoclonal Antibody||100 μL||$239|
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