WWE Affinity Resin Set

N° du produit ABIN1741736

Détail du produit

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

Type d'anticorp: Recombinant

Reactivité: Humain

Hôte: Escherichia coli (E. coli)

Conjugué: GST tag

Application: Pull-Down Assay (Pull-Down)

Specificité: 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.

Attributs du produit: Set contains one ABIN1741734 and ABIN1741735.

Purification: Affinity chromatography

Pureté: > 95 %

Matériel non inclus: 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

Information d'application

Indications d'application: 20 µL=20 µg per reaction (for each)

Commentaires:

280.00

Protocole: 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.

Restrictions: For Research Use only

Stockage

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

Agent conservateur: Sodium azide

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

Conseil sur la manipulation: Do not freeze!

Stock: 4 °C

Stockage commentaire: Stable for 6 months from date of shipment when stored at 4 ˚C.

Référence

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

Détail du antigène

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

Autre désignation: RNF146 (RNF146 Produits)

Synonymes: 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

Sujet: 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.

Poids moléculaire: 8 kDa + GST