RFP-Trap® M

ABIN509412 产品详细信息, 供应商: Log in to see
抗原
适用
Discosoma
6
1
1
宿主
Camelidae
抗体类型
Recombinant Antibody
标记
Magnetic Particles
应用范围
Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Immunoprecipitation (IP), Mass Spectrometry (MS), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
选项
Supplier
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原理 RFP-Trap® is a high quality RFP-binding protein coupled to a monovalent matrix (magnetic particles) for biochemical analysis of RFP fusion proteins and their interacting partners.
品牌 RFP-Trap®
样品类型 Cell Extracts
特异性 Binding capacity: 10 µL RFP-Trap®_M slurry binds 0.25 - 0.5 µg of RFP
交叉反应 (详细) RFP-Trap efficiently pulls down various Red fluorescent proteins derived from DsRed, e.g. mRFP1, mCherry, mOrange, mPlum but also mRuby, mKate2 and RFP-tagged fusion proteins. No cross-reaction to DsRed, mRFPruby, TagRFP and all GFPs can be detected.
产品特性 Antibodies - extremely powerful tools in biomedical research - are large complex molecules (~ 150 kDa) consisting of two heavy and two light chains. Due to their complex structure, the use of antibodies is often limited and hindered by batch-to-batch variations.

Camelidae (camels, dromedaries, llamas and alpacas) possess functional antibodies devoid of light chains, so-called heavy chain antibodies (hcAbs). hcAbs recognize and bind their antigens via a single variable domain (VHH). These VHH domains are the smallest intact antigen binding fragments (~ 13 kDa).

Nano-Traps are based on single domain antibody fragments (VHHs) derived from alpaca.
组件 RFP-Trap® coupled to magnetic particles
试剂未包括 Lysis buffer (CoIP), 10x RIPA buffer, Dilution buffer, Wash buffer, Elution buffer
别名 RFP (RFP ELISA Kit 摘要)
研究领域 Tags/Labels
应用备注 Red fluorescent proteins (RFP) and variants thereof are widely used to study protein localization and dynamics. For biochemical analyses including mass spectroscopy and enzyme activity measurements these RFP-fusion proteins and their interacting factors can be isolated fast and efficiently (one step) via immunoprecipitation using the RFP-Trap. The RFPTrap_A enables purification of any protein of interest fused to RFP.
说明

Bead size 0.5 - 1 µm

实验时间 1.5 h
实验流程
  • Robust and versatile tool for biochemical analyses of RFP-fusion proteins
  • Short incubation times (5 - 30 min)
  • Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
  • Low unspecifi c binding
  • No contaminating heavy and light chains of conventional antibodies
  • Applicable in Chromatin Immunoprecipitation (ChIP)
试剂准备

Suggested buffer composition

  • Lysis buffer (CoIP): 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA,0.5% NP-40
  • 10x RIPA buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, 1% Deoxycholate
  • Dilution buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
  • Wash buffer: 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA
  • Elution buffer: 200 mM glycine pH 2.5

实验流程

Before you start: Add 1ml PBS to your cells and scrape them off the petri dish.Transfer to precooled tube, spin 3 min at 500 x g and discard supernatant. Wash cell pellet twice with ice cold PBS, briefly resuspending the cells.

  • 1. For one immunoprecipitation reaction resuspend cell pellet (~10^7 mammalian cells) in 200 µL lysis buffer by pipetting (or using a syringe).
    optional: add 1 mM PMSF and Protease inhibitor cocktail (not included) to lysis buffer
    optional for nuclear/chromatin proteins: add 1 mg/ml DNase and 2.5 mM MgCl2 (not included) to lysis buffer
  • 2. Place the tube on ice for 30 min with extensively pipetting every 10 min.
  • 3. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C.
  • 4. Transfer supernatant to a pre-cooled tube. Adjust volume with dilution buffer to 500 µL – 1000 µL. Discard pellet.
    optional: add 1 mM PMSF and Protease inhibitor cocktail (not included) to dilution buffer
    note: the cell lysate can be frozen at this point for long-term storage at -80°C

For immunoblot analysis dilute 50 µL cell lysate with 50 µL 2x SDS-sample buffer (à refer to as input).
  • 5. Equilibrate RFP-Trap®_M beads in dilution buffer. Resuspend magnetic beads by vortexing and transfer 20 - 30 µL bead slurry in 500 µL ice cold dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and wash beads 2 more times with 500 µL ice cold dilution buffer.
  • 6. Add cell lysate to equilibrated RFP-Trap®_M beads and incubate the RFPTrap®_M beads with the cell lysate under constant mixing for 10 min – 2 h at room temperature or 4°C.
    note: during incubation of protein sample with the RFP-Trap®_M the final concentration of detergents should not exceed 0.2% to avoid unspecific binding to the matrix.
  • 7. Magnetically separate beads until supernatant is clear. For western blot analysis dilute 50 µL supernatant with 50 µL 2x SDS-sample buffer (à refer to as nonbound). Discard remaining supernatant.
  • 8. Wash beads three times with 500 µL ice cold wash buffer. After the last wash step, transfer beads to new tube.
    optional: increase salt concentration in the second washing step up to 500 mM
  • 9. Resuspend RFP-Trap®_M beads in 100 µL 2x SDS-Sample buffer or go to step 11.
  • 10. Boil resuspended beads for 10 minutes at 95°C to dissociate the immunocomplexes from the beads. The beads can be magnetically separated and SDS-PAGE is performed with the supernatant (à refer to as bound).
  • 11. optional: elute bound proteins by adding 50 µL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by centrifugation. Transfer the supernatant to a fresh cup and add 5 µL 1M Tris base (pH 10.4) for neutralization. To increase elution efficiency this step can be repeated.

限制 仅限研究用
浓度 2.5 mL resin
缓冲液 1 x PBS,0.01% Sodium azide
储存液 Sodium azide
注意事项 This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
注意事项 Do not freeze.
储存条件 4 °C
有效期 12 months
厂商提供的图像
 image for RFP-Trap® M (ABIN509412) RFP-Trap A
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