WWE Affinity Resin Set

Produktnummer ABIN1741736

Produktdetails

Target: RNF146 (Ring Finger Protein 146 (RNF146))

Antikörpertyp: Recombinant

Reaktivität: Human

Wirt: Escherichia coli (E. coli)

Konjugat: GST tag

Applikation: Pull-Down Assay (Pull-Down)

Spezifität: The WWE Affinity Resin, ABIN1741734 is highly purified GST-RNF146(100-175) fusion protein construct expressed in E. coli, and bound to glutathione beads.
WWE Negative Control Resin, ABIN1741735 is identical to the ABIN1741734 resin except for R163A substitution, which effectively abolishes PAR binding. The negative control resin is useful to control for non-specific binding, and its use is optional.
Both products will be delivered each in 0.5 mL (0.5 mg fusion protein) supplied as a slurry containing approx. 50 µL resin.

Produktmerkmale: Set contains one ABIN1741734 and ABIN1741735.

Aufreinigung: Affinity chromatography

Reinheit: > 95 %

Benötigtes Material: Lysis buffer (e.g.: 50 mM Tris, pH 8, 200 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 10 % glycerol, 1 mM DTT, 0.1 % SDS, and protease inhibitors)
Cell/tissue extract containing approx. 0.15 to 1 mg total protein per sample
Microcentrifuge tubes
Microcentrifuge
SDS-PAGE sample buffer

Anwendungsinformationen

Applikationshinweise: 20 µL=20 µg per reaction (for each)

Kommentare:

280.00

Protokoll: 1. Resuspend the WWE affinity and neg control resins by gently inverting the product tubes several times to obtain a homogenous suspension of resin.
2. Use a wide-bore pipette or a cut pipette tip to transfer 20 µL of the suspension to approx. 0.5 mL of lysis buffer in a Microfuge tube.
3. Sediment resin at 10k x g in a Microfuge for 20 s. Carefully remove most of the lysis buffer, leaving the resin (barely visible) undisturbed in the tube. NOTE: Position the tubes in the Microfuge with the hinge oriented outward in order to ascertain the location of the sedimented resin.
4. Add cell/tissue extract in lysis buffer to the Microfuge tube containing the resin. Suggested extract protein amount is 0.15 to 1 mg in a total buffer volume of 0.5 mL.
5. Incubate the reaction for several hours or overnight at 4 °C on a Nutator or similar device.
6. Sediment, then wash resin 3-times with 0.5-1 mL lysis buffer, as in step 3. On the final wash, carefully remove residual buffer without disturbing the resin.
7. Add 75 µL 1X SDS-PAGE sample buffer to each tube, agitate, then incubate at 95 °C for 10 min to dissociate GST-macrodomain from PARylated proteins and the resin.
8. Run samples on SDS-PAGE, and perform Western blotting. Probe immunoblot using desired protein-specific antibodies, for example anti-PARP1 (ABIN1741708) to detect affinity purified proteins. Compare results to negative control resin samples to assess non-specific binding, which should be minimal.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Format: Liquid

Buffer: 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, and 0.02 % sodium azide

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handhabung: Do not freeze!

Lagerung: 4 °C

Informationen zur Lagerung: Stable for 6 months from date of shipment when stored at 4 ˚C.

Referenzen

Zhong, Yeh, Hao, Pourtabatabaei, Mahata, Shao, Chessler, Chi: "Nutritional energy stimulates NAD+ production to promote tankyrase-mediated PARsylation in insulinoma cells." in: PLoS ONE, Vol. 10, Issue 4, pp. e0122948, (2015) (PubMed).

Antigendetails

Target: RNF146 (Ring Finger Protein 146 (RNF146))

Andere Bezeichnung: RNF146 (RNF146 Produkte)

Synonyme: RP3-351K20.1 Accessory Reagents, dJ351K20.1 Accessory Reagents, 2610509H23Rik Accessory Reagents, Iduna Accessory Reagents, RNF146 Accessory Reagents, rnf146 Accessory Reagents, dactylidin Accessory Reagents, wu:fi29e08 Accessory Reagents, zgc:55909 Accessory Reagents, ring finger protein 146 Accessory Reagents, RING finger protein 146 Accessory Reagents, ring finger protein 146 S homeolog Accessory Reagents, RNF146 Accessory Reagents, Rnf146 Accessory Reagents, rn146 Accessory Reagents, rnf146.S Accessory Reagents, rnf146 Accessory Reagents

Sub Type: Cocktail

Hintergrund: RNF146 (Iduna) is a RING-domain E3 ubiquitin ligase that positively regulates Wnt signalling. RNF146 directly interacts with poly(ADP-ribose) through its WWE domain. The WWE domain is a conserved globular domain found in multiple PARPs and E3 ligases.

Molekulargewicht: 8 kDa + GST