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Isotype controls should be concentration matched and run alongside the primary antibody samples. Analyze cells in DNA staining solution on flow cytometer. Electrotransfer to nitrocellulose membrane ATP 10 mM for kinase assays: This datasheef be done by re-exposing the blot to ECL reagents and making sure there is no datwsheet prior to adding the next primary antibody.

Place tube back datasheer magnetic separation rack. Proceed to immunoprecipitation section. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube.

Resuspend cells in 0. Aspirate media from cultures; wash cells with 1X PBS; aspirate. Microcentrifuge for 5 min. Find answers on our FAQs page. This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase daatasheet utilizing Protein A magnetic separation.


Western blot analysis of extracts from various cell lines using USP8 Antibody. Solutions and Reagents From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: Incubate 30 min on ice. Antibodies are purified by protein A and peptide affinity chromatography. Scrape cells off the plate and transfer to microcentrifuge tubes.

Phospho-STING (Ser366) (D8K6H) Rabbit mAb #40818

Biotinylated Protein Ladder Detection Pack: Find answers on our FAQs page. Incubate with rotation for 20 min at room temperature. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS. Aliquot desired number of cells into tubes or wells.

Reprobing of an existing membrane datsheet a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. To Purchase S View sizes. Allow cells to fix for 15 min at room temperature.

Discard supernatant in appropriate waste container. Incubate for 30 min at room temperature.

Repeat washing step once more. Immunoprecipitation for Native Proteins This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.

Proceed with Immunostaining Section C. Transfer supernatant containing phosphorylated substrate to another tube. Wash by centrifugation in incubation buffer. Adjust pH to 8. Carefully remove the buffer once the solution is clear.


Research studies have shown that USP8 is an essential growth-regulated enzyme indespensible for cell proliferation and survival 3,4. Dilute to 1X with dH 2 O. Sonicate for 10—15 sec to complete cell lysis and shear DNA to reduce sample viscosity.

Datasheet, PDF – Free Datasheets, GHz Fixed Modulus Dividers

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro of human USP8 protein. Fix datasheeh 15 min at room temperature.

Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1—2 hr at room temperature in the dark. Additionally, it is dagasheet that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment.

Remove buffer once solution is clear.

Immunoprecipitation Cell Lysate Pre-Clearing Optional A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Proceed with detection Section D.

Recommended Anti-Rabbit secondary antibodies: