Product overview

Name Anti-GFP antibody ValidAbTM
Description

Antibody 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.

Host Rabbit
Clonality Polyclonal
Target GFP
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Validation data

Figure 1. pEGFP-C2 transfected HEK293 cells showing co-localised staining of EGFP and HB8912.

Signals derived from EGFP and HB8912 completely overlap showing 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. HB8912 was incubated overnight (4°C) at a 1:1000 dilution (1µg/ml) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35561, 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 SPE confocal laser scanning microscope coupled to a Leica DMi8 inverted epifluorescence microscope. The image was captured using a 63x objective, 405nm (29.0% power), 488nm (37.9% power) and 532nm (36.8% power) laser lines in a z-stack (0.28 µm spacing). 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 2. Specific HB8912 staining only in pEGFP-C2 transfected HEK293 cells.

Figure 2. Specific HB8912 staining only in pEGFP-C2 transfected HEK293 cells. HB8912 revealed a primary 31.5kDa 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 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 130V for 120 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 HB8912 (Polyclonal rabbit anti-GFP) at a 1:10,000 dilution (0.1µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) 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 (15g/l glycine, 1g/l SDS, 1% Tween 20, pH2.2) before being washed, blocked for 2 hours in 5% non-fat dry milk and incubated in HB9177 (1:4,000 dilution, 0.25µg/ml) overnight at 4°C. Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-mouse HRP conjugated, Sigma Aldrich A3682) for 2hrs 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 3. Concentration response of HB8912 staining in pEGFP-C2 transfected HEK293 cells.

HB8912 shows consistent results at dilutions as low as 1:64,000 (15.6 ng/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 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 130V for 120 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 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB8912. Each strip was incubated separately with a separate HB8912 concentration with this ranging from 2µg/ml (1:500 dilution) to 7.8ng/ml (1:128,000 dilution). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) 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 4. The effect of varying HB8912 concentration upon staining in pEGFP-C2 transfected HEK293 cells.

HB8912 produced excellent amplification of native EGFP signal at concentrations as low as 0.5µg/ml (1:2000 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. HB8912 was incubated overnight (4°C) at concentrations ranging from 0.5 to 4µg/ml (1:2000 to 1:250) followed by a one hour incubation with secondary antibody (Polyclonal goat anti-rabbit DyLight 594 conjugated, Thermofisher 35561, 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 DMi8 inverted epifluorescence microscope (20x objective) coupled to a Leica DFC365FX monochrome digital camera with DAPI LP, FITC LP and RHOD_LP filters. Exposure times were as follows: 


  • 1:250    DAPI LP: 1x gain, 49.8ms exposure; FITC LP: 2.5x gain, 142.3ms exposure; RHOD LP: 2x gain, 108.8ms exposure
  • 1:500    DAPI LP: 1x gain, 49.8ms exposure; FITC LP: 1.2x gain, 117.2ms exposure; RHOD LP: 1.2x gain, 108.8ms exposure
  • 1:1000    DAPI LP: 1x gain, 49.8ms exposure; FITC LP: 1.4x gain, 101.3ms exposure; RHOD LP: 1.2x gain, 246.0ms exposure
  • 1:2000    DAPI LP: 1x gain, 49.8ms exposure; FITC LP: 1.4x gain, 86.6ms exposure; RHOD LP: 1.7x gain, 246.0ms exposure.


Images were processed in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682) using the subtract background (50px rolling ball radius) tool followed by stacking and montage creation. 

Figure 5. EGFP tagged Cannabinoid receptor 1 c-terminal domain (EGFP-ctCB1R) detected in virally transfected HEK293 cells using HB8912.

HB8912 succesfully detected the shift in molecular weights when HEK293 cells were either transfected with a EGFP-ctCB1R construct or EGFP alone. Method: Sindbis virus was used to over-express EGFP-ctCB1R alongside EGFP control in HEK293 cells following the cited methodology (Fletcher Jones, 2020. PhD Thesis, University of Bristol). After allowing time for expression, lysates were prepared and loaded (equal loading) alongside a protein ladder (New England Biolabs, P7719S) onto a 10% acrylamide gel. The cytosol fraction from rat brains was also loaded as a non EGFP expressing control and was prepared following established methodology (Molnar et al., 1993. Neuroscience 53:307-326). Gels were 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 HB8912 (Polyclonal rabbit anti-GFP) at a 1:1,000 dilution (1µg/ml). Following washing the membrane was incubated in secondary antibody (1:10,000 dilution, Polyclonal goat anti-rabbit HRP conjugated, Sigma Aldrich A6154) 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).

Product information

Immunogen full length EGFP protein
Purification Affinity purification using immunogen as ligand
Concentration 1mg/ml
Formulation Lyophilised. When reconstituted contains PBS with 15mM sodium azide and 1% recombinant BSA
Predicted species reactivity NA
Tested species reactivity NA

Tested applications

Applications ICC, WB
Western blot optimal concentration

Dependent upon sample GFP expression. We used 100ng/ml (1:10,000 dilution) in pEGFP-C2 transfected HEK293 cells.

ICC optimal concentration Dependent upon sample GFP expression. We used as low as 500ng/ml (1:2,000 dilution) in pEGFP-C2 transfected HEK293 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 with HB8912 recognising these.

Storage & Handling

Storage instructions

-20°C then use reconstitution advice

Reconstitution advice

We recommend reconstituting with either:


  • dH2O and storing at 4°C
  • 50:50 ratio of dH2O to glycerol and storing at -20°C
  • dH2O then aliquot and store at -80°C


Take care when opening as the precipitate is extremely light and can easily be lost if disturbed. When reconstituting make sure that the antibody is thoroughly dissolved by pipetting up and down before giving the antibody a brief spin at <10,000g to make sure that all material is recovered and at the bottom of the tube.


For more information please see our detailed guide on storing and using your antibody

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

References for Anti-GFP antibody ValidAbTM

References are publications that support the biological activity of the product

FAQs

Does this GFP antibody react with both GFP and EGFP?

This GFP antibody has been reported to recognise both GFP and its enhanced format

Does this GFP antibody react with YFP?

This GFP antibody has been reported to recognise EYFP protein although we have not independently tested this.

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