Anti-GFP抗体(Sepharose) (ab69314)

概述

  • 产品名称Anti-GFP抗体(Sepharose)
    参阅全部 GFP 一抗
  • 描述
    兔多克隆抗体to GFP (Sepharose)
  • 偶联物Sepharose
  • 特异性This product is a suspension of affinity purified anti-GFP antibody (ab6556) covalently linked to sepharose beads. It is reactive against all variants of Aequorea victoria GFP such as S65T-GFP, RS-GFP, YFP, CFP, RFP, and EGFP. The unit sold contains 25µg of affinity purified rabbit anti-GFP IgG cross-linked to 125 µl sepharose beads in a total volume of 250 µl buffer. The product is supplied as a 50% slurry to facilitate pipetting. Pipetting the slurry is facilitated when pipet tips are blunted by cutting 3-5mm off from the tip.
  • 经测试应用适用于: IPmore details
  • 种属反应性
    Not applicable.
  • 免疫原

    Recombinant full length protein corresponding to GFP.

  • 常规说明This product should be kept refrigerated at all times whilst in short term storage. Using sterilised equipment will reduce the risk of bacterial contamination.

性能

  • 形式Liquid
  • 存放说明Shipped at 4°C. Store at +4°C. Do Not Freeze.
  • 存储溶液Preservative: 0.05% Sodium Azide
    Constituents: PBS
  • Concentration information loading...
  • 纯度Immunogen affinity purified
  • 纯化说明This antibody is an affinity purified rabbit anti-GFP antibody purified on an affinity chromatography column made with highly purified recombinant GFP.
  • 克隆多克隆
  • 同种型IgG
  • 研究领域

应用

Our Abpromise guarantee covers the use of ab69314 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
IP Use at an assay dependent concentration.

We rountinely recommend using 20 µL of slurry per IP, which corresponds to 2 µg of IgG cross-linked to beads. this results in a clearly visible 10 µL bead pellet upon centrifugation. Unconjugated version: ab6556. FITC conjugated version: ab66180. Biotin conjugated version: ab69313.

靶标

  • 相关性Function: Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca2+ -activated photoprotein aequorin.

    Subunit structure: Monomer.

    Tissue specificity: Photocytes.

    Post-translational modification: Contains a chromophore consisting of modified amino acid residues. The chromophore is formed by autocatalytic backbone condensation between Ser-65 and Gly-67, and oxidation of Tyr-66 to didehydrotyrosine. Maturation of the chromophore requires nothing other than molecular oxygen.

    Biotechnological use: Green fluorescent protein has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors. Fluorescent proteins and its mutated allelic forms, blue, cyan and yellow have become a useful and ubiquitous tool for making chimeric proteins, where they function as a fluorescent protein tag. Typically they tolerate N- and C-terminal fusion to a broad variety of proteins. They have been expressed in most known cell types and are used as a noninvasive fluorescent marker in living cells and organisms. They enable a wide range of applications where they have functioned as a cell lineage tracer, reporter of gene expression, or as a measure of protein-protein interactions. Can also be used as a molecular thermometer, allowing accurate temperature measurements in fluids. The measurement process relies on the detection of the blinking of GFP using fluorescence correlation spectroscopy.

    Sequence similarities: Belongs to the GFP family.

    Biophysicochemical properties: Absorption: Abs(max)=395 nm
    Exhibits a smaller absorbance peak at 470 nm. The fluorescence emission spectrum peaks at 509 nm with a shoulder at 540 nm.
  • 别名
    • GFP antibody
    • Green fluorescent protein antibody
    • yfp antibody

Anti-GFP antibody (Sepharose) 图像

  • Lane 1 & 3: IP from COS 7 cells transfected with EGFP.N-Ras (48 kDa)

    Lane 2 & 4: IP from untransfected COS 7 cells

    Lane 1 & 2: IP using 15 ul of rabbit anti-GFP conjugated to sepharose beads (0.5 mg IgG per ml of beads)

    Lane 3 & 4: IP using 15 ul of goat anti-GFP conjugated to sepharose beads (1 mg IgG per ml of beads)

    Immunoprecipitation: COS 7 cell lysates containing 100 ug of total protein in 200 ul of 0.1% SDS-RIPA buffer with addition of complete protease inhibitor were used for each immunoprecipitation. Cell lysates were incubated with anti-GFP sepharose beads for 2 hours at 4oC with rocking. Beads were washed. Proteins were eluted with 1% SDS 50 mM HEPES (pH 7.4) at 80oC (15 min). Half of the IP sample was loaded on each lane of 10 % SDS PAGE gel and gel was processed for Western blotting/ECL.

    Western blot: Primary antibody: monoclonal mouse anti-GFP (ab291) at 0.2 ug/ml in 5% non-fat milk/TBS-T; 1 hour incubation at room temperature. Secondary antibody: Sheep anti-mouse IgG HRP conjugate at 1/20,000 dilution in 5% non-fat milk/TBS-T; 1 hour incubation at room temperature. Development with ECL plus (GE Healthcare)

    Exposure time: 5 seconds

Anti-GFP antibody (Sepharose) (ab69314)参考文献

This product has been referenced in:
  • Diamond MI  et al. Subcellular localization and Ser-137 phosphorylation regulate tumor-suppressive activity of profilin-1. J Biol Chem 290:9075-86 (2015). Read more (PubMed: 25681442) »
  • Jia L  et al. A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation. J Biol Chem 289:6249-57 (2014). IP . Read more (PubMed: 24385443) »

See all 7 Publications for this product

Product Wall

Due to the very high titer, assume near stochiometric binding: 1 nmole antibody will bind 2 nmole GFP. For example, 150ug antibody (since M.W. 150kDa) will bind 50ug GFP (since GFP M.W. ˜25kDa and IgG have two Fabs).

Application Immunoprecipitation
Immuno-precipitation step Other - The product itself has the antibody attached to the sepharose beads
Sample Saccharomyces cerevisiae Cell lysate - whole cell (yeast cell lysate)
Specification yeast cell lysate
Total protein in input 5 µg
Username

Abcam user community

Verified customer

提交于 Mar 26 2014



We typically recommend using 20ul of slurry per IP. Taking into account this volume as well as the number of IPs you expect to conduct at any one time should give you a good idea of what volumes to make your aliquots.

The exact protocol will vary and depends on the expression level of the GFP fusion and the type of protein and the purpose of the experiment.

The full protocol can be found in Megan C. Yap*#, Morris A. Kostiuk*, Dale D. O. Martin, Maneka A. ...

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To our knowledge, this antibody has not been specifically tested for co-IP of plant proteins. It will, however, be guaranteed for that purpose as long as GFP is expressed in your plant material. The only references we are currently aware of that use th...

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The beads can appear to be translucent white when in the slurry. This is normal.Before taking out beads from the stock solution, it is important to mix the slurry well.

After the IP, when pelleting the bead-antibody-protein complex, the pelle...

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I recommend to test-run a smaller sample first. I can recommend to use 20ulslurry (2ug of IgG cross-linked beads)with 100ug protein and check the result. If this works optimal with your samples, I then can recommend to scale it up.

Since 3mg ...

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Replies to your questions: 1. 10ul Beads (20ul slurry) will contain 1ug IgG, each IgG (MW:150,000) can bind 2 GFP molecules (~MW:25,000 each ), therefore about 666ng of GFP should bind to these beads. Cross-linking should not interfere too much with t...

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Thank you for your inquiry. Both antibodies will work quite well actually, but we would recommend the antibody cross-linked to sepharose-beads (ab69314) simply because the lab worked with it for a much longer period of time than the antibody cross-l...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"