GFP-Catcher (agarose magnetic beads)

N° du produit ABIN7272855

Détail du produit

Highlights:

• High-affinity anti-GFP single-domain antibody (sdAb) covalently bound to 4 % cross-linked agarose magnetic beads with 50-150 µm diameter.
• The Alpaca sdAb clone 1H1 is specific for GFP and many GFP derivatives. Capacity >4 µg GFP / 1 µL packed beads (2 µL bead slurry).
• Compatible with physiological buffers, common Lysis and Wash Buffers, and high stringency buffers.

Antigène: GFP (Green Fluorescent Protein (GFP))

Reactivité: Aequorea victoria

Hôte: Alpaca

Expression System: E.coli

Application: Protein Complex Immunoprecipitation (Co-IP), Immunoprecipitation (IP), Purification (Purif), Chromatin Immunoprecipitation (ChIP), RNA-Binding Protein Immunoprecipitation (RIP)

Fonction: GFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4% cross-linked magnetic agarose.

Specificité: GFP (green fuorescent protein) and common GFP derivatives like EGFP, mEGFP, Sirius, tSapphire, Cerulean, eCFP, mTurquoise, acGFP, Emerald, superecliptic pHluorin, paGFP, superfolder GFP, eYFP, mVenus and Citrine. Other not tested.

Aucune reactivité croisée: Does not cross-react with mCherry, dsRed, mRFP, mTagBFP or their most common derivatives.

Attributs du produit: GFP-Catcher is based on a high-affinity single-domain antibody (sdAb) that is covalently immobilized on 4 % cross-linked magnetic agarose beads. The innovative, oriented and selective attachment via a flexible linker guarantees a high accessibility of the sdAbs and largely eliminates batch-to-batch variations. Due to the single-chain nature of sdAbs and their covalent attachment, no "leakage" of light and heavy chains from IgGs is observed during elution with SDS sample buffer.
GFP-Catcher thus features high affinity and superior capacity for GFP fusion proteins while showing negligible non-specific background.
GFP-Catcher immobilizes a wide range of GFP derivatives.
GFP-Catcher is compatible not only with physiological buffers but also with high stringency buffers.
GFP-Catcher thus provides great freedom to adjust the binding and washing conditions to the experimental needs.

Matériel non inclus: wash buffers, columns, tubes

Bead Ligand: Antibody

Bead Matrix: Magnetic Agarose beads

Bead Size: 90 μm

Informations sur le produit:

Notre GFP Catcher est un produit basé sur la technique GFP Pull-Down et utilisé pour isoler les protéines de fusion de la GFP à partir de lysats cellulaires. Il se compose de billes magnétiques spéciales (produit ABIN7272855) ou de billes d'agarose (produit ABIN5311508) recouvertes d'un anticorps contre la GFP. Ces billes permettent une liaison efficace et sélective à la GFP ou aux protéines cibles fusionnées à la GFP.

L'utilisation de GFP Catcher est similaire à celle de la méthode GFP Pull-Down. Tout d'abord, la protéine à étudier est exprimée dans les cellules, alors qu'elle est fusionnée avec la GFP. Ensuite, le lysat cellulaire est préparé pour extraire les protéines cibles. Les billes de capture de la GFP sont alors placées dans le lysat cellulaire et liées à ces protéines par une liaison spécifique à la GFP ou à la protéine de fusion de la GFP.

Une fois que les billes contenant les protéines cibles liées ont été isolées, une purification minutieuse est effectuée afin d'éliminer les composants non spécifiquement liés. Enfin, les protéines cibles peuvent être séparées des billes et utilisées pour d'autres analyses telles que l'immunoblotting ou la spectrométrie de masse.

GFP Catcher offre l'avantage d'isoler rapidement et facilement les protéines de fusion GFP. C'est un outil utile pour l'analyse des protéines et il permet d'identifier efficacement les protéines qui interagissent avec la protéine de fusion GFP. GFP Catcher est souvent utilisé dans la recherche en biologie cellulaire pour étudier les interactions protéine-protéine et approfondir la compréhension des processus cellulaires.

Information d'application

Commentaires:

4% cross-linked magnetic agarose (bead size 50-150 μm) with covalently immobilized single-domain antibody

Protocole:

This protocol provides a general outline of how to use GFP-Catcher (agarose magnetic beads) for immunoprecipitation using a microcentrifuge for sedimentation. Alternatively, it is possible to use GFP-Catcher agarose beads in spin columns. All protocol steps should be carried out at 4 °C.

1. For mammalian cells, harvest 10⁶-10⁸ cells per sample.
2. Lyse cells according to established protocols in 0.2 to 1.5 mL volume. Recommended Buffer Conditions: GFP-Catcher resins are compatible with commonly used Lysis and Washing buffers, e.g. RIPA buffer. The following buffer conditions have been tested:
   • pH ranging from pH 5 to pH 9
   • 2 % Triton X-100, 1 % Tween-20, 1 % NP-40, 1 % CHAPS, 1 % Deoxycholate, 0.1 % SDS
   • 4 M NaCl, 2 M KCl, 1 M MgCl₂
   • 100 mM EDTA
   • 4 M urea
   • 10 mM DTT, 10 mM 2-Mercaptoethanol
   • Protease Inhibitors
   • RNAse A, DNAse I, Benzonase
3. Centrifuge cell lysates in microcentrifuge tubes for 10 min at 14.000 x g at 4 °C. Keep a small samples as “input” fraction.
4. Transfer the supernatant to a fresh microcentrifuge tube for each sample and keep at 4 °C.
5. Homogenize the GFP-Catcher (agarose magnetic beads) slurry gently by shaking.
6. Transfer 20 μL bead slurry to a 1.5 mL microcentrifuge tube for each sample.
7. Add 1 mL Lysis Buffer to equilibrate GFP-Catcher (agarose magnetic beads).
8. Place the tubes on a magnet stand until the fluid is clear. Remove the supernatant carefully.
9. Repeat wash steps once for a total of two washes.
10. Resuspend equilibrated GFP-Catcher (agarose magnetic beads) gently with the cell lysate supernatant.
11. Rotate the microcentrifuge tubes for 1 h at 4 °C.
12. Place the tubes on a magnet stand until the fluid is clear. Keep a small aliquot of the supernatant as “unbound” fraction. Carefully remove the supernatant.
13. Resuspend GFP-Catcher (agarose magnetic beads) in 1 mL Lysis Buffer.
14. Place the tubes on a magnet stand until the fluid is clear and carefully remove the supernatant.
15. Repeat wash steps twice for a total of three washes.
16. Resuspend GFP-Catcher (agarose magnetic beads) gently in 1 mL TBS.
17. Place the tubes on a magnet stand until the fluid is clear and carefully remove the supernatant.
18. Repeat wash steps once for a total of two washes.
19. Resuspend GFP-Catcher (agarose magnetic beads) resin in 50 µL 2X SDS samples buffer.
20. Heat GFP-Catcher (agarose magnetic beads) resin for 5 min to 95 °C.
21. Place the tubes on a magnet stand until the fluid is clear and transfer the supernatant to fresh microcentrifuge tubes. Keep the GFP-Catcher (agarose magnetic beads) as backup.

Restrictions: For Research Use only

Stockage

Buffer: 50 % slurry in PBS containing 20 % Ethanol

Stock: 4 °C

Stockage commentaire: Store at 4°C for up to 12 months. Do not freeze!

Date de péremption: 12 months

Détail du antigène

Antigène: GFP (Green Fluorescent Protein (GFP))

Autre désignation: GFP (GFP Produits)

Synonymes: green fluorescent protein Bead, gfp Bead