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RFP-Trap® MA Kit

ABIN2452226 产品详细信息, 供应商: Log in to see
抗原
适用
Discosoma
6
1
1
宿主
Camelid (Camelidae)
抗体类型
Recombinant Antibody
标记
Magnetic Agarose Beads
应用范围
Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Mass Spectrometry (MS), Immunoprecipitation (IP), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
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原理 RFP-Trap® is a high quality RFP-binding protein coupled to a monovalent matrix (magnetic agarose beads) 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®_MA slurry binds 3 - 4 μg of RFP
交叉反应 (详细) tested on RFP, mCherry, mOrange, mPlum, tagRFP
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 GFP-Trap® coupled to magnetic agarose beads
Lysis buffer (CoIP) 40 mL
10x RIPA buffer 2 mL
Dilution buffer 150 mL
Wash buffer 40 mL
Elution buffer 3 mL
别名 RFP
背景 RFP fluorescent proteins (RFPs) and variants thereof are widely used to study proteinlocalization and dynamics. For biochemical analysis including mass spectrometry and enzyme activity measurements these RFP-fusion proteins and their interacting factors can be isolated fast and efficiently by immunoprecipitation using the RFP-Trap®. RFP-Trap® utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose beads.
研究领域 Tags/Labels
应用备注 Optimal working dilution should be determined by the investigator.
说明

Particle size ~ 40 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 unspecific binding
- No contaminating heavy and light chains of conventional antibodies
- Applicable in Chromatin Immunoprecipitation (ChIP)
样品收集 Harvest cells:
For one immunoprecipitation reaction the use of 10^6 - 10^7 mammalian cells (approx. one 10 cm dish) expressing a RFP-tagged protein of interest is recommended. To harvest adherent cells, aspirate growth medium, add 1 mLice-cold PBS to cells and scrape cells from dish. Transfer cells to a pre-cooled tube, spin at 500 g for 3 min at +4 °C and discard supernatant. Wash cell pellet twice with ice-cold PBS, gently resuspending the cells.

Lyse cells
1. Resuspend cell pellet in 200 μL ice-cold lysis buffer by pipetting or using a syringe.
note: Supplement lysis buffer with protease inhibitors and 1 mM PMSF (not included). optional for nuclear/chromatin proteins: Use RIPA buffer supplemented with 1 mg/mL DNase, 2.5 mM MgCl2, protease inhibitors and 1 mM PMSF (not included).

2. Place the tube on ice for 30 min with extensively pipetting every 10 min.

3. Centrifuge cell lysate at 20.000x g for 10 min at +4 °C. Transfer lysate to a pre-cooled tube. Add 300 μL dilution buffer to lysate. Discard pellet.
note: At this point cell lysate may be put at -80 °C for long-term storage. optional: Add 1 mM PMSF and protease inhibitors (not included) to dilution buffer
We recommend that during incubation with the beads the final concentration of detergents does not exceed 0.2 % to avoid unspecific binding to the matrix. If required, use more dilution buffer to dilute the supernatant accordingly.
实验流程

Equilibrate beads
4. Vortex RFP-Trap®_MA beads and pipette 25 μL bead slurry into 500 μL ice-cold dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.
Bind proteins:
5. Add diluted lysate (step 3) to equilibrated RFP-Trap®_MA beads (step 4). If required, save 50 μL of diluted lysate for immunoblot analysis. Tumble end-over-end for 1 hour at 4 °C.
6. Magnetically separate beads until supernatant is clear. If required, save 50 μL supernatant for immunoblot analysis. Discard remaining supernatant.
Wash beads:
7. Resuspend RFP-Trap®_MA beads in 500 μL dilution buffer. Magnetically separate beads until supernatant is clear. Discard supernatant and repeat wash twice.
optional: Increase salt concentration in the second washing step up to 500 mM.
Elute proteins:
8. Resuspend RFP-Trap®_MA beads in 100 μL 2x SDS-sample buffer.
9. Boil resuspended RFP-Trap®_MA beads for 10 min at 95 °C to dissociate immunocomplexes from RFP-Trap®_MA beads. The beads can be magnetically separated and SDS-PAGE is performed with the supernatant.
10. optional instead of steps 8 and 9: elute bound proteins by adding 50 μL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by magnetic separation. Transfer the supernatant to a new tube 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
缓冲液 Storage buffer: 20 % EtOH
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® MA Kit (ABIN2452226) RFP-Trap® MA Kit
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