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Signals derived from mCherry and HB6512 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 pmCherry-C3 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. HB6512 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 488 conjugated, Thermofisher 35552, 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 (35.7% power), 488nm (35.7% power) and 532nm (36.8% power) laser lines in a z-stack (0.35 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). |
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Figure 2. Specific HB6512 staining only in pmCherry-C3 transfected HEK293 cells. HB6512 revealed a primary 31.9kDa band only present in pmCherry-C3 transfected cells without any cross-reactivity with EGFP 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 HB6512 (Polyclonal rabbit anti-mCherry) 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). |
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Figure 3. The effect of varying HB6512 concentration upon staining in pmCherry-C3 transfected HEK293 cells. HB6512 produced excellent amplification of native mCherry 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 pmCherry-C3 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. HB6512 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 488 conjugated, Thermofisher 35552, 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, 61.0ms exposure; FITC LP: 2.1x gain, 152.5ms exposure; RHOD LP: 4.5x gain, 521.8ms exposure
1:500 DAPI LP: 1x gain, 61.0ms exposure; FITC LP: 3.2x gain, 202.0ms exposure; RHOD LP: 3.7x gain, 488.7ms exposure
1:1000 DAPI LP: 1x gain, 61.0ms exposure; FITC LP: 3.2x gain, 394.8ms exposure; RHOD LP: 3.7x gain, 224.3ms exposure
1:2000 DAPI LP: 1x gain, 61.0ms exposure; FITC LP: 3.2x gain, 350.1ms exposure; RHOD LP: 2.8x gain, 297.1ms 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. |
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Figure 4. Concentration response of HB6512 staining in pmCherry-C3 transfected HEK293 cells. HB6512 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 pmCherry-C3 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 HB6512. Each strip was incubated separately with a separate HB6512 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. |
