Validation Report #029688 Validation Date: 04/29/14

Summary

Antigen mCherry

Catalog number ABIN1760652

Supplier St John's Laboratory

Supplier catalognumber STJ34373

Lot number Not specified

Method validated Western Blot

Laboratory Shakti Bioresearch LLC

Validation number 029688

Positive ControlmCherry-transfected HeLa cells 24, 48 and72 hours post transfection

Negative Control Untransfected HeLa cells (0 hours)

NotesA strong clear band was observed in thepositive control sample, and not in thenegative control sample.

Independent Results

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

Full MethodsPrimary Antibody

Antigen: mCherryCatalog number: ABIN1760652Supplier: St John's LaboratorySupplier catalog number: STJ34373Lot number: Not specified

Loading Control AntibodyAntibody: ActinSupplier: Cell Signaling TechnologiesCatalog number: CST 4967Lot number: 7

Secondary AntibodyAntibody: Goat anti-mouse HRP conjugateSupplier: Bio-RadCatalog number: 170-5047Lot number: HA 101954

ControlsPositive 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 TechnologiesCatalog 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 (CellSignaling Technologies Catalog No. 9803) with protease inhibitors from Roche.

ProtocolLysates 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 weightstandard.

Protein samples were transferred to nitrocellulose membrane using iBlot gel transfer apparatus (Invitrogen CatalogNo. 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 NotesNo challenges noted.No non-specific bands observed.