Poly-ADP-ribose Affinity Resin Set

Produktnummer ABIN1741733

Produktdetails

Target: Af1521 Macrodomain

Antikörpertyp: Recombinant

Reaktivität: Archaeoglobus fulgidus

Wirt: Escherichia coli (E. coli)

Konjugat: GST tag

Applikation: Pull-Down Assay (Pull-Down)

Spezifität: The PAR Affinity Resin, ABIN1741731 is highly purified GST-Af1521 macrodomain fusion protein construct expressed in E. coli, and bound to glutathione beads. The Af1521 macrodomain protein is reported to also bind mono-ADP-ribosylated proteins and ADP-ribose.
PAR Negative Control Resin, Cat. ABIN1741732 is identical to the ABIN1741731 resin except for two gly to asp substitutions, which abolish PAR binding. The negative control resin is useful to control for non-specific binding, and its use is optional.
Both products are supplied as 1 mL slurry containing approx. 75 µL resin (1 mg fusion protein)

Produktmerkmale: Set contains one ABIN1741731 and ABIN1741732.

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, 0.5 % deoxycholate, 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 PAR 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. 1 mL of lysis buffer in a Microfuge tube.
3. Sediment resin at 15k x g in a Microfuge (highest speed setting) for 20 s. Carefully remove most of the lysis buffer, leaving the resin (barely visible) undisturbed in the tube.
NOTE: Position 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), or anti-poly-ADP-ribose antibodies (ABIN1741702 or ABIN1741707) 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: PBS with 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

Gagné, Pic, Isabelle, Krietsch, Ethier, Paquet, Kelly, Boutin, Moon, Foster, Poirier: "Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress." in: Nucleic acids research, Vol. 40, Issue 16, pp. 7788-805, (2012) (PubMed).

Antigendetails

Target: Af1521 Macrodomain

Sub Type: Cocktail

Hintergrund: Af1521 is a thermopohilic protein from Archaeoglobus fulgidus, and contains a conserved approx. 190 AA domain known as the macro domain. Macrodomains are found in a wide variety of organisms including bacteria, viruses, and vertebrates. Expressed and purified macrodomains from Af1521, Alc1, macroH2A and Bal/PARP9 proteins have been shown to bind polymeric ADP-ribose modified proteins with high specificity and affinity

Molekulargewicht: 21 kDa + GST