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Appropriate datashest controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Ubiquitinating enzymes UBEs catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme DUB action 1,2.

If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

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Pre-wash magnetic beads just prior to use: Count cells using a hemocytometer or alternative method. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser of human STING protein. A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Western blot analysis of extracts from various cell lines using USP8 Antibody.

Sonicate for 10—15 sec to complete cell lysis and shear DNA to reduce sample viscosity. Incubate 30 min on ice. Incubate with rotation for 20 min at room temperature.


Wash cells by centrifugation in excess 1X PBS to remove methanol. Aliquot desired number of cells into tubes or wells. Keep on ice between washes. ATP 10 mM for kinase assays: Prepare solutions with reverse osmosis deionized RODI or equivalent grade water. Mix well then add 0. Do not aliquot the antibody. 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.

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Reprobing of an existing membrane is a convenient means to immunoblot for datasheet proteins independently when only a limited amount of sample is available. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Aspirate fixative, rinse three times in 1X PBS for 5 min each. Would you like to visit your country specific website? To Purchase S View sizes. Would you like to visit your country specific website? Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

Isotype controls should be concentration matched and run alongside the primary antibody samples. Aspirate media from cultures; wash cells with 1X PBS; aspirate. Incubate with rotation for 20 minutes datqsheet room temperature.

Incubate for 1 hr at room temperature. Dilute to 1X with dH 2 O. Sample Analysis Proceed to one of the following specific set of steps. Do not aliquot the antibody.


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Find answers on our FAQs page. Protein A Magnetic Beads: Wash by centrifugation in incubation buffer. To harvest cells dataheet nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.

Incubate substrate with membrane for 1 minute, remove excess solution membrane remains wetwrap in plastic and expose to X-ray film. Repeat washing step once more.

Pre-clear enough lysate for test samples and isotype controls. USP8 Antibody – To Purchase S View sizes. The optimal lysate concentration will depend on the expression level of the protein of interest. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.

Primary Antibody Dilution Buffer: Remove buffer once solution is clear. Place the tube in a magnetic separation rack for seconds. Proceed to analyze by western immunoblotting or kinase activity section D.

Fix for 15 min at room temperature.