Anti-GFP antibody ValidAb™

(HB6381)
Technical documents: Datasheet

Product overview

Name Anti-GFP antibody ValidAb™
Host Mouse
Clonality Monoclonal
Target GFP
Description

Monoclonal antibody (IgM) to GFP - green coloured fluorescent protein widely used as a tag in molecular biology. Part of the ValidAb™ range of highly validated, data-rich antibodies. 

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Validation data

Figure 1. Specific HB6381 staining only in pEGFP-C2 transfected HEK293 cells.

HB6381 revealed a 30.7kDa band only present in pEGFP-C2 transfected cells without any cross-reactivity with mCherry or native proteins. Method: HEK293 cells were cultured and transfected following established protocols (Lee et al., 2019. PLoS ONE, 14(5):e0213116) with either pEGFP-C2 or pmCherry-C3 plasmids. After allowing 3 days for expression, lysates were prepared (see our guide on WB sample preparation) and loaded (equal loading) onto a 15% acrylamide gel alongside a protein ladder (New England Biolabs, P7719S) before being run at 60V for 30 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6381 at a 1:8,000 dilution (125ng/ml). Following washing, the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Following imaging the membrane was stripped with two changes of stripping buffer (HB7756) before being washed, blocked for 1 hour in 5% non-fat dry milk and incubated in HB9177 (mouse monoclonal anti-GAPDH, 1:4,000 dilution, 250ng/ml) and HB7863 (mouse monoclonal anti-HSP60, 1:5,000 dilution, 200ng/ml) for 2 hours at room temperature. Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 1 hour and visualised again using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure).

Figure 2. Concentration response of HB6381 staining in pEGFP-C2 transfected HEK293 cells.

HB6381 shows consistent results at dilutions as low as 1:128,000 (7.8ng/ml). Method: HEK293 cells were cultured and transfected following established protocols (Lee et al., 2019. PLoS ONE, 14(5):e0213116) with a pEGFP-C2 plasmid. After allowing 3 days for expression, lysate was prepared (see our guide on WB sample preparation) and loaded (equal loading) onto a 15% acrylamide gel alongside a protein ladder (New England Biolabs, P7719S) before being run at 60V for 30 minutes followed by 120V for 100 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 3hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6381. Each strip was incubated separately with a separate HB6381 concentration with this ranging from 500ng/ml (1:1000 dilution) to 1.95ng/ml (1:512,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs. For more detail please see our Western blotting protocol. Detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 10 min exposure, 700nm channel: 30 sec exposure). Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.

Figure 3. pEGFP-C2 transfected HEK293T cells showing co-localised staining of EGFP and HB6381.

Signals derived from EGFP and HB6381 overlap showing strong evidence for specificity. Method: HEK293 cells were cultured and transfected following established protocols (Lee et al., 2019. PLoS ONE, 14(5):e0213116) with a pEGFP-C2 plasmid. After allowing cells time to express, fixation was carried out using 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6381 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 594 conjugated, Thermofisher 35511, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope attached to a Photometric-Prime95B camera. Images were captured as a z-stack using a 40x objective, L5 (8ms exposure) and TX2 (4.1ms exposure) filters. Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 4. Concentration response of HB6381 staining in pEGFP-C2 transfected HEK293T.

HB6381 shows strong staining at concentrations as low as 125ng/ml (1:8,000 dilution). Method: HEK293 cells were cultured and transfected following established protocols (Lee et al., 2019. PLoS ONE, 14(5):e0213116) with a pEGFP-C2 plasmid. After allowing cells time to express, fixation was carried out using 4% PFA. Cells were permeabilised with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB6381 was incubated overnight (4°C) at dilutions ranging from 1:1,000 to 1:8,000 (0.125-1µg/ml). This was followed by a one hour incubation with secondary antibody (Polyclonal goat anti-mouse DyLight 594 conjugated, Thermofisher 35511, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope attached to a Photometric-Prime95B camera. Images were captured using a 40x objective as a z-stack using the following exposure settings:
  • 1:1,000; L5: 8ms, TX2: 4.1ms
  • 1:2,000: L5: 30ms, TX2: 4.1ms
  • 1:4,000: L5: 5ms, TX2: 8ms
  • 1:8,000: L5: 5ms, TX2: 9ms
Deconvolution was carried out using Huygens Essential (Scientific Volume Imagine) followed by the stack being flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Product information

Immunogen

Recombinant prot-r-AcGFP expressed in and purified from E. coli

Epitope Localised to the N-terminus of both GFP (amino acids 1-17) and recombinant prot-r-AcGFP (amino acids 36-53) to the sequence MVSKGAELFTGIVPILIE
Clone number 1F1
Isotype IgM
Purification

Protein L affinity chromatography

Concentration 1 mg/ml
Formulation 50% PBS, 50% glycerol + 5mM sodium azide
Predicted species reactivity Species Independent
Tested species reactivity Species Independent

Tested applications

Applications ICC, WB
Western blot optimal concentration

Dependent upon sample GFP expression. We used 125ng/ml (1:8,000 dilution) in pEGFP-C2 transfected HEK293 cells.

ICC optimal concentration

Dependent upon sample GFP expression. We used 500ng/ml (1:2,000 dilution) in pEGFP-C2 transfected HEK293T cells.

Positive control

Any tissue or cell sample that has been engineered to express GFP.

Negative control

Any wild type tissue or cellular sample.

Open data link

Please follow this this link to OSF

Target information

Other names

EGFP, green fluorescent protein, EYFP

UniProt ID P42212
Structure image  Chemical Structure
Gene name GFP
NCBI full gene name green fluorescent protein
Amino acids

238 (27kDa)

Isoforms

None

Expression

Exogenously expressed only. Not expressed natively in mammalian cells.

Subcellular expression

GFP is generally expressed cytosolically in basic constructs however expression can be directed to any cellular compartment through GFP-tagged proteins that naturally express in only certain compartments.

Processing

NA

Post translational modifications

NA

Homology (compared to human)

NA

Similar proteins

EGFP (enhanced GFP, 26.9kDa) and YFP (yellow fluorescent protein, 26.4kDa) are both extremely similar.

Epitope homology (between species)

NA

Epitope homology (other proteins)

In a BLAST search considering potential cross-reactivities with human, rat and mouse proteins the following proteins were identified:

  • Bromodomain-containing protein 3 (Human) - 100% identity across 38% of the query
  • NADH-ubiquinone oxidoreductase chain 1 (Human) - 100% identity across 33% of the query
  • Tudor domain containing protein 6 (Human) - 80% identity across 50% of the query
  • Sodium/hydrogen exchanger 11 (Human) - 80% identity across 55% of the query.

 

However none of these cross-reactivites were observed experimentally implying that the short query covers were insufficient to produce immunoreactivity to non-GFP epitopes.

Storage & Handling

Storage instructions

-20°C

Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use

FAQs

What mounting media do you recommend to use with this antibody?
Does this GFP antibody cross-react with mCherry?

We have tested and found no cross-reactivity between this GFP antibody and mCherry in Western blot experiments.

What guarantee do you have that my GFP antibody will perform as expected?

We guarantee that your GFP antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.

What protocols are available for use with this GFP antibody

We have made a comprehensive collection of protocols that we have used in our experiments to validate this GFP antibody.

What counterstains do you recommend for use in ICC and IHC with this GFP antibody?

We recommend using either DAPI or Hoechst 33342 to label cell nuclei. In some experiments it is also helpful to label actin filaments in the cytoskeleton using a Phalloidin conjugate such as FITC Phalloidin or Rhodamine Phalloidin-TRITC.

Any other questions?

For any other questions about our antibody products please see our technical FAQs for antibodies

References for Anti-GFP antibody ValidAb™

References are publications that support the biological activity of the product
  • Green fluorescent protein: a perspective.

    Remington SJ (2011) Protein science : a publication of the Protein Society 20 : 1509-19
  • Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes.

    Stepanenko OV et al (2008) Current protein & peptide science 9 : 338-69
  • A guide to choosing fluorescent proteins.

    Shaner NC et al (2005) Nature methods 2 : 905-9
  • The green fluorescent protein.

    Tsien RY (1998) Annual review of biochemistry 67 : 509-44
  • Crystal structure of the Aequorea victoria green fluorescent protein.

    Ormö M et al (1996) Science (New York, N.Y.) 273 : 1392-5