Recombinant A. victoria GFP protein (ab84191)

Reviews (3)Q&A (5)References (1)

Overview

Description

  • Nature
    Recombinant
  • Source
    Escherichia coli
  • Amino Acid Sequence
    • Accession
      P42212
    • Species
      Aequorea victoria
    • Sequence
      MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTFSYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT HGMDELYK
    • Molecular weight
      27 kDa
    • Amino acids
      1 to 238

Specifications

Our Abpromise guarantee covers the use of ab84191 in the following tested applications.

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

  • Applications

    Functional Studies

    SDS-PAGE

    Western blot

  • Endotoxin level
    < 1.000 Eu/µg
  • Mass spectrometry
    MALDI-TOF
  • Purity
    > 95 % SDS-PAGE.
    Purified using conventional chromatography techniques.
  • Form
    Liquid
  • Additional notes

    Endotoxin Level determined by LAL method

  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

    pH: 8.00
    Constituents: 0.316% Tris HCl, 10% Glycerol

    This product is an active protein and may elicit a biological response in vivo, handle with caution.

General Info

  • Alternative names
    • GFP
    • Green fluorescent protein
  • Relevance
    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.

Images

  • SDS-PAGE - Recombinant <em>A. victoria</em> GFP protein (ab84191)
    SDS-PAGE - Recombinant A. victoria GFP protein (ab84191)

    15% SDS-PAGE showing ab84191 at approximately 26.8kDa (3µg).

  • Functional Studies - Recombinant <em>A. victoria</em> GFP protein (ab84191)
    Functional Studies - Recombinant A. victoria GFP protein (ab84191)

    1. Prepare a 150 μl recombinant GFP with various concentrations (0.00074 nM - 6.21 μM) in assay buffer and equilibrate to 25°C. (Assay buffer: 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.02% sodium azide.)

      2. Read at excitation wavelengths 485 nm and emission 535 nm.

    - 96 Well Polystyrene Microplate, black

    - Fluorescent plate reader

References

This product has been referenced in:
  • Shiota M  et al. Gold-nanofève surface-enhanced Raman spectroscopy visualizes hypotaurine as a robust anti-oxidant consumed in cancer survival. Nat Commun 9:1561 (2018). Read more (PubMed: 29674746) »
See 1 Publication for this product

Publishing research using ab84191? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-8 of 8 Abreviews or Q&A

Purified GFP for Western Blot

 Excellent
Abreviews
abreview image
Application
Western blot
We have used this protein for Western blot applications. On a 4-12% Bis-Tris gel, the protein runs to the expected molecular weight and is readily detectable using multiple anti-GFP antibodies in our hands. We observed excellent signal linearity across a large range of GFP amounts (as shown in the image) by Western blot and have not observed loss of protein or change in apparent molecular weight following long term storage at -20 C.

Dr. Sam Nowitzki

Verified customer

Submitted Jun 29 2015

Western blot standard

 Excellent
Abreviews
Application
Western blot
We use this as a positive standard in our western blots to detect expressed GFP fusion proteins in HEK cells.

Abcam user community

Verified customer

Submitted Dec 17 2014

Western blot abreview for A. victoria GFP full length protein

 Excellent
Abreviews
Application
Western blot
Used the protein as a positive control in Western Blot of a GFP-tagged recombinant protein preparation. Result: good specific signal, no nonspecific bands detected.

Block: TBS + 3 % milk 30 min.
Antibody incubation: HRP-conjucated goat polyclonal to GFP (Abcam ab6663) in 1:2000 dilution, 60 min.
Wash: 4 x 5 min TBS-T.
Detection: 5 min incubation in TMB Liquid Substrate System (Sigma).

Abcam user community

Verified customer

Submitted Dec 08 2014

Question

Does ab84191 have a his-tag or any other tag?

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Answer

I can confirm ab84191 is not a tagged protein.

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Question

looking for purified RFP and GFP protein
RFP: only ab51993
GFP: ab84191 - what does active mean? Does protein fluoresce the same way (spectra) as if it was expressed in cells/tissue?
ab116434 - Does protein fluoresce the same way (spectra) as if it was expressed in cells/tissue?
difference between ab84191 and ab116434?

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Answer

Attached is the result of the lab's excitation and emission test. They don’t have an excitation and emission curve, but on their equipment the excitation wavelength was 485nm and emission 535nm.
They haven’t verified it’s optimal peak, but there will be variations from one ELISA reader to another and there should be a certain amount of flexibility.


The protein is listed as "active" because we tested excitation and emission. Although we haven’t compared our recombinant protein with cell/tissue expressed GFP, in principle it should fluoresce the same way (spectra).


Both GFP protein have two different sources. The protein sequence is the same, but the validation has been done differently.

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Question

I see this protein is stored in a Tris based buffer. Would the protein still be functional if stored in a PBS based buffer?
Also, I need to label the GFP protein via primary amines. Would there be lysines available on GFP to accomplish this labeling?

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Answer

Unfortunately we haven't tested this in PBS and haven’t tried to label the GFP protein via primary amines, so we cannot confirm whether there will be lysines available on GFP to accomplish this labeling.

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Question

what mass ofGFP protein is being measured in the fluorescence graph, where GFP emmission is measured against uM GFP?

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Answer

The molecular weight of GFP is 27 kDa. A 1.0 uM solution of GFP contains 27 micrograms of GFP per milliliter of solution. In this assay, 200 ul is added to each well, so for the 1 uM standard, the well contains 5.4 ug, and for the 2 uM standard, 10.8 ug, and so on.

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Question

I am interested in the above product: GFP protein (Active). Before I raise a PO, may I ask if GFP is stable under mild oxidative conditions, e.g., 0.06% H2O2. I notice that there are cysteine residues within the sequence of GFP and my main fear is the cysteines forming disulfide bonds? Is that a possibility? I intend to load your GFP into a hydrogel matrix which also contains cysteines residues and I am also concern that cysteines in GFP will form di-sulfide bonds with the cysteines in my matrix. Essentially, I do not want any covalent interactions between GFP and matrix under the mild oxidative environment mentioned above. Any suggestions? Cheers! Best wishes

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Answer

In regards to the exposure to the laboratory has regrettably not verified the stability of this protein under these mild oxidative conditions, e.g., 0.06% H2O2. Depending on the exposure time the answer would probably vary also. I am sorry therefore to say, that we can not provide a clear answer to this question. In regards to the di'sulifde bonds in hydrogel, indeed the laboratory also thinks that it is possible for disulfide bonds to form with cys 48 (cys 70 is buried within the core of the protein). Perhaps using DTT or other reducing agents can work.

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