製品の概要

  • 製品名
    Anti-GFP antibody (Sepharose)
    GFP 一次抗体 製品一覧
  • 製品の詳細
    Rabbit polyclonal to GFP (Sepharose)
  • 由来種
    Rabbit
  • 標識
    Sepharose
  • 特異性
    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.
  • アプリケーション
    適用あり: IPmore details
  • 種交差性
    Not applicable.
  • 免疫原

    Recombinant full length protein corresponding to GFP.
    Database link: P42212

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

製品の特性

  • 製品の状態
    Liquid
  • 保存方法
    Shipped at 4°C. Store at +4°C. Do Not Freeze.
  • バッファー
    Preservative: 0.05% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • 精製度
    Immunogen affinity purified
  • 特記事項(精製)
    This antibody is an affinity purified rabbit anti-GFP antibody purified on an affinity chromatography column made with highly purified recombinant GFP.
  • ポリ/モノ
    ポリクローナル
  • アイソタイプ
    IgG
  • 研究分野

アプリケーション

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.

アプリケーション Abreviews 特記事項
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.

ターゲット情報

  • 関連性
    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.
  • 別名
    • GFP antibody
    • Green fluorescent protein antibody

画像

  • 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

プロトコール

参考文献

This product has been referenced in:
  • Chou YT  et al. Identification of danthron as an isoform-specific inhibitor of HEME OXYGENASE-1/cytochrome P450 reductase interaction with anti-tumor activity. J Biomed Sci 25:6 (2018). Human . Read more (PubMed: 29361943) »
  • Jeong SI  et al. XAF1 forms a positive feedback loop with IRF-1 to drive apoptotic stress response and suppress tumorigenesis. Cell Death Dis 9:806 (2018). Read more (PubMed: 30042418) »
See all 19 Publications for this product

レビューと Q&A

1-10 of 10 Abreviews or Q&A

Question
Answer

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).

Read More
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

Abcam user community

Verified customer

投稿 Mar 26 2014

Answer



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.

Read More

Answer

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. Perinpanayagam, Pieter G. Hak, Anjaiah Siddam, Janaki R. Majjigapu, Gurram Rajaiah, Bernd O. Keller, Jennifer A. Prescher, Peng Wu, Carolyn R. Bertozzi, John R. Falck and Luc G. Berthiaume (2010) “Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry” J. Lipid Res. 51:1566-80.

Adapted from Yap et al. above...
Immunoprecipitation
Cells ( 1 °— 10 6 to 1 °— 10 7 ) were washed with cold PBS, harvested, and lysed with cold EDTA-free RIPA buffer by rocking for 15 min at 4°C. Cell lysates were centrifuged at 16,000 g for 10 min at 4°C and the postnuclear supernatants were collected.
[0.1% SDS, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium-
deoxycholate, 2 mM MgCl 2 , EDTA-free complete protease inhibitor]
GFP fusion proteins were immunoprecipitated from approximately 1 mg of protein lysates (depending on the expression level of the GFP fusion larger amount of lysate may be needed) with affinity purifi ed goat anti-GFP cross-linked to Sepharose beads by rocking for 2 h or overnight at 4°C. ...the rabbit anti-GFP-sepharose sold by Abcam can also be used.
The beads were extensively washed with 0.1% SDS-RIPA, resuspended
in 50 mM HEPES, pH 7.4, with 1% SDS containing buffer or any standard SDS-PAGE loading buffer depepnding on the application or purpuse of the experiment, and heated for 15 min at 80°C (or 5 min at 95C or 2min at 100C (again depending on the application).
. The supernatants containing the fusion proteins were collected.
Please note that this would be a guideline only and may require some individual optiization.

Read More

Question
Answer

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 this antibody are:

Serio G et al. Small GTPase Rab5 participates in chromosome congression and regulates localization of the centromere-associated protein CENP-F to kinetochores. Proc Natl Acad Sci U S A 108:17337-42 (2011). IP. PubMed: 21987812

Carmena A et al. The Rap1-Rgl-Ral signaling network regulates neuroblast cortical polarity and spindle orientation. J Cell Biol 195:553-62 (2011). IP. PubMed: 22084305

Read More

Answer

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 pellet should be clearly visible and stable so that the supernatant can be taken off without any bigger difficulties.

Please let me know how the results is looking on the WB. I am happy to help if optimisation is need.

Read More

Answer

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 would mean 600ul slurry, I can recommend to optimise further. Maybe 250ul slurry will be enough (this is one vial of ab69314).

I am sorry I do not have a definitive answer to this question. This has not been experimentally tested by us and we therefore cannot recommend any definitive amounts.

I can confirm that elution of the protein after IP can be done by boiling in loading buffer. This will most probably also detach some of the IP antibody from the beads (althoughantibody and beads are covalently bound)and therefore there is the risk of getting heavy and light chains of the IP antibody into the gel.

Please check with your MALDI-TOF protocol for requirements on IP and gel run. .

Read More

Answer

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 the binding. 2. Proteins were eluted with 1% SDS 50 mM HEPES (pH 7.4) at 80oC (15 min). 3. The cross-link was carried out by a proprietary process that should minimize leaking of both IgG heavy and light chains. Typically less than 5% of the IgG content on the beads will leak. 4. By boiling samples there is a risk of having the antibody eluted too, therefore a mild eluent, such as glycine buffer, should be used to avoid this from happening.

Read More

Question
Answer

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-linked to magnetic beads (ab69315). I would recommend our rabbit-anti-GFP-crosslinked to sepharose beads for the IP, then use Ab290 or Ab6556 for the WB. If the GFP concentration is really low, you may want to increase the volume of the sample by making more of it. Our IP protocol worked just as well in 1ml or 10 ml because of the high avidity of the antibody for the antigen. Incubate sample with the antibodies for 2-4 hours at 4C. Keep the amount of beads low and centrifuge thoroughly to increase beads recovery. If you incubate overnight make sure you have protease inhibitors in the lysates. I hope this information helps. Please contact me with any other questions.

Read More

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

登録