mCherry is a fluorophore used in biotechnology as a tracer to follow the flow of fluids and as a marker when tagged to molecules and cell components. mCherry and the majority of red fluorescent proteins derive from a protein isolated from Discosoma sp. mCherry is sometimes preferred to other fluorophores due to its colour, as well as its photostability compared to other monomeric fluorophores. St John’s Laboratory supplies a highly photostable mCherry tag antibody, which is resistant to photobleaching. To purchase this antibody, use the following link: http://www.stjohnslabs.com/mcherry-tag-antibody?filter_name=STJ34373

1. Validation Report #029688 Validation Date: 04/29/14
Summary
Antigen mCherry
Catalog number ABIN1760652
Supplier St John's Laboratory
Supplier catalog
number
STJ34373
Lot number Not specified
Method validated Western Blot
Laboratory Shakti Bioresearch LLC
Validation number 029688
Positive Control
mCherry-transfected HeLa cells 24, 48 and
72 hours post transfection
Negative Control Untransfected HeLa cells (0 hours)
Notes
A strong clear band was observed in the
positive control sample, and not in the
negative control sample.

2. Independent Results
Figure 1: Western blot of HeLa cell lysate 24, 48 and 72 hours after
transfection with mCherry. MW, molecular weight markers. 0,
untransfected HeLa cell lysate. Actin, loading control.

3. Full Methods
Primary Antibody
Antigen: mCherry
Catalog number: ABIN1760652
Supplier: St John's Laboratory
Supplier catalog number: STJ34373
Lot number: Not specified
Loading Control Antibody
Antibody: Actin
Supplier: Cell Signaling Technologies
Catalog number: CST 4967
Lot number: 7
Secondary Antibody
Antibody: Goat anti-mouse HRP conjugate
Supplier: Bio-Rad
Catalog number: 170-5047
Lot number: HA 101954
Controls
Positive control: HeLa cells were grown to 70% confluency in a 6-well plate and transfected with 1 µg of
mCHERRY encoding plasmid DNA (Addgene plasmid #36084) using DMRIE-C tranfecting reagent (Catalog No.
10459-014, lot No. 890718 from Invitrogen). Cell lysates were made in 1X lysis buffer (Cell Signaling Technologies
Catalog No. 9803) with protease inhibitors from Roche 24, 48 and 72 hours after transfection.
Negative control: HeLa cells were grown to 80% confluency and cell lysates were made in 1X lysis buffer (Cell
Signaling Technologies Catalog No. 9803) with protease inhibitors from Roche.
Protocol
Lysates were mixed with NuPAGE® LDS Sample Buffer (Life Technologies NP0007) with 10 mM DTT and
denatured for 5 min at 90ºC.
10 µg of each lysate was electrophoresed on a NuPAGE® Novex® 4-12% Bis-Tris Gel (Life Technologies
NP0322BOX) and run in NuPAGE® MOPS SDS Running Buffer (Life Technologies NP0001) at 100 volts (30 mA)
for 2.5 h.
Precision plus protein dual color standards (BIO-RAD Catalog No. 161-0374) was run as a molecular weight
standard.
Protein samples were transferred to nitrocellulose membrane using iBlot gel transfer apparatus (Invitrogen Catalog
No. IB301002)
The membrane was blocked in 1 x TBS-T + 5% Milk for 2 h at RT.
The membrane was washed for 3 x 10 min in 1 x TBS-T.
The membrane was incubated with the primary antibody diluted 1:500 in 1 x TBS-T + 0.5% BSA and incubated
overnight at 4°C.
The membrane was washed 3 x 10 min in 1 x TBS-T.
The membrane was incubated with mouse HRP secondary antibody diluted 1:10,000 in 1 x TBS-T + 0.5% BSA
and incubated for 60 min at RT.
The membrane was washed for 3 x 10 min in 1 x TBS-T.
Proteins were detected using ECL detection (Thermo Scientific 34075) and visualized with x-ray film.
Detection exposure time was 5 min.
Experimental Notes
No challenges noted.
No non-specific bands observed.