Anti-GFP antibody ValidAb™

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).

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.

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).

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).