Product overview

Name Anti-mCherry Antibody ValidAbTM
Description

Antibody to mCherry - red coloured fluorescent protein widely used as a tag in molecular biology

Host Rabbit
Clonality Polyclonal
Target mCherry
Write Your Own Review
You're reviewing:Anti-mCherry Antibody ValidAb<sup>TM</sup>
Rate this item:

Validation data

Figure 1. pmCherry-C3 transfected HEK293 cells showing co-localised staining of mCherry and HB6512.

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).

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).

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. 

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.

Product information

Immunogen Recombinantly expressed full-length mCherry protein
Purification Affinity chromatography using immunogen as ligand
Concentration 1mg/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 mCherry expression. We used 100ng/ml (1:10,000 dilution) in pmCherry-C3 transfected HEK293 cells.
ICC optimal concentration Dependent upon sample mCherry expression. We used 500ng/ml (1:2,000 dilution) in pmCherry-C3 transfected HEK293 cells.
Positive control

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

Negative control

Any wild type tissue or cellular sample.

Open data link

Please follow this link to OSF

Target information

Other names Pamcherry
UniProt ID D1MPT3
Structure image  Chemical Structure
Gene name PAmCherry
Amino acids 236 (26.8kDa)
Isoforms None
Expression Exogenously expressed only. Not natively expressed in mammalian cells.
Subcellular expression mCherry is generally expressed in the cytosol however expression can be directed towards any cellular compartment through mCherry-tagged fusion proteins that traffick to specific compartments.
Processing NA
Post translational modifications

NA

Similar proteins None

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

References for Anti-mCherry Antibody ValidAbTM

References are publications that support the biological activity of the product
  • Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

    Heppert J et al (2016) Mol Biol Cell 27(22) : 3385-3394
  • A guide to choosing fluorescent proteins

    Shaner N, Steinbach P and Tsien R (2005) Nature Methods 2(12) : 905-909
  • Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

    Shaner N et al (2004) Nature Biotechnology 22(12) : 1567-1572
  • Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed)

    Bevis B and Glick B (2002) Nature Biotechnology 20(11) : 1159

FAQs

Any other questions?

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