Anti-GFP antibody ValidAbTM
(HB8912)
Product overview
| Name | Anti-GFP antibody ValidAbTM |
| Host | Rabbit |
| Clonality | Polyclonal |
| Target | GFP |
| Customer comments | The GFP antibody shows good specificity and signal/noise (S/N). At equivalent dilution, the signal is brighter with this antibody than with our usual antibodies - the Poncer lab, Institute Du Fer À Moulin - Inserm. |
| 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. |
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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 (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 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 (HB7756) 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 (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 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 (see our guide on WB sample preparation) 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).
Figure 6. Comparison of HB8912 and Millipore MAB377 staining in GFP expressing neurons within mouse brain slices
"Immunostainings comparing GFP staining in mouse brain slices. The GFP antibody shows good specificity and signal/noise (S/N). At equivalent dilution, the signal is brighter with this antibody than with our usual antibodies"
Data kindly provided by the Poncer lab, Institute Du Fer À Moulin - Inserm
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 (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 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 (HB7756) 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 (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 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 (see our guide on WB sample preparation) 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).
Figure 6. Comparison of HB8912 and Millipore MAB377 staining in GFP expressing neurons within mouse brain slices
"Immunostainings comparing GFP staining in mouse brain slices. The GFP antibody shows good specificity and signal/noise (S/N). At equivalent dilution, the signal is brighter with this antibody than with our usual antibodies"
Data kindly provided by the Poncer lab, Institute Du Fer À Moulin - Inserm
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 | Species Independent |
| Tested species reactivity | Species Independent |
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 |  |
| 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 | Upon receipt store at either -20°C or -80°C.
For 100μg packs either:
For 25μg packs either:
For more information read our guide on the best care for your product. 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. |
| Important | This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use |
Technical guides
Western Blot Protocol (1 MB) Immunocytochemistry Protocol (1.2 MB) Immunohistochemistry Protocol (1.6 MB) References for Anti-GFP antibody ValidAbTM
References are publications that support the biological activity of the product
-
Green fluorescent protein: A perspective
Remington SJ (2011) Protein Science 20(9) : 1509-1519 -
Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes
Stepaneko O et al (2008) Curr Protein Pept Sci. 9(4) : 338-369 -
A guide to choosing fluorescent proteins
Shaner N, Steinbach P and Tsien R (2005) Nature Methods 2(12) : 905-909 -
The green fluorescent protein
Tsien RY (1998) Annu Rev Biochem 67 : 509-544 -
Crystal structure of the Aequorea victoria green fluorescent protein
Ormö M et al (1996) Science 273(5280) : 1392-1395
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