ProLite™ Orange Protein Gel Stain 5000X

Three-fold dilution series of BSA standards were separated on a NuPAGE® 4–12% Bis-Tris gel and stained with A) ProLite™ Orange Protein Gel Stain or B) Coomassie brilliant blue (CBB) according to standard protocols. The ProLite™ Orange stained gels were photographed using a SYPRO Orange filter. The CBB-stained gels were photographed using transmitted white light without an optical filter.<br />Lane 1: 15ug, Lane 7: ~20ng, Lane 10: ~0.8 ng BSA.

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	486.72
Solvent	Water

Spectral properties

Excitation (nm)	484
Emission (nm)	586

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Molecular weight

486.72

Excitation (nm)

484

Emission (nm)

586

Prolite™ Orange is an alternative protein stain that can be used to replace SYPRO Orange Protein Gel Stain (SYPRO is the trademark of ThermoFisher). Prolite™ Orange is a sensitive, ready-to-use fluorescent stain for total protein detection in 1D gels. The sensitivity of Prolite™ Orange is as good as or better than traditional silver staining techniques. Stained proteins can be viewed with a standard UV or blue-light transilluminator or imaging equipment containing the appropriate filters or lasers. Fluorescent stains are rapid, and highly sensitive for detecting total protein in protein electrophoresis gels and membranes. The Prolite™ Orange fluorescent stain can be used for total protein quantitation and can be viewed using a standard UV or blue-light transilluminator or with imaging instruments equipped with appropriate light sources.

Example protocol

AT A GLANCE

Storage and Handling

Store at -20 °C protected from light. Product is stable for at least 12 months from the date of receipt when stored as recommended. The ProLite™ Orange diluted in acetic acid or buffer can be stored in glass or plastic bottles at 4 °C for three months, protected from light. Before opening, each vial should be allowed to warm to room temperature and then briefly centrifuged in a microfuge to deposit the DMSO solution at the bottom of the vial. If dye particles are present, briefly sonicate the tube or vortex the tube vigorously.

Safety

We advise researchers to follow universal laboratory safety precautions when handling ProLite™ Orange dye.

PREPARATION OF WORKING SOLUTION

ProLite™ Orange solution (5000X)

Dilute the 5000X ProLite™ Orange stock solution to make 1X ProLite™ Orange stock solution using 7.5% (v/v) acetic acid and mix vigorously.
Note The working solution can be reused up to four times. However, we observed significant reduction in response after the second reuse. It’s highly recommended to use fresh working solution for the optimal result.

SAMPLE EXPERIMENTAL PROTOCOL

The following protocol is recommended and can be used as guideline. However some comparisons might be needed to determine which one better meets your needs.

Staining Proteins after Electrophoresis

Run gels as per your protocols.
Pour the working solution into a small plastic dish.
Note For one to two standard size minigels, use about 50 mL of working solution. For larger gels, use between 500 to 700 mL of working solution.
Note Make sure to add enough working to completely immerse the gels.
Place the gel into the working solution.
Note Cover the container with aluminum foil to protect the dye from light.
Gently agitate the gel at room temperature for 10 to 60 minutes.
Rinse briefly with 7.5% acetic acid.
Gels may be visualized on a standard 300 nm UV transilluminator or with a blue-light transilluminator.
Destaining: Gels can be mostly destained by incubation overnight in 0.1% Tween® 20. Alternatively, incubation in several changes of 7.5% acetic acid will eventually remove all of the stain.

Calculators

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of ProLite™ Orange Protein Gel Stain *5000X* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	205.457 µL	1.027 mL	2.055 mL	10.273 mL	20.546 mL
5 mM	41.091 µL	205.457 µL	410.914 µL	2.055 mL	4.109 mL
10 mM	20.546 µL	102.728 µL	205.457 µL	1.027 mL	2.055 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

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Spectral properties

Excitation (nm)	484
Emission (nm)	586

Images

Figure 1. Three-fold dilution series of BSA standards were separated on a NuPAGE® 4–12% Bis-Tris gel and stained with A) ProLite™ Orange Protein Gel Stain or B) Coomassie brilliant blue (CBB) according to standard protocols. The ProLite™ Orange stained gels were photographed using a SYPRO Orange filter. The CBB-stained gels were photographed using transmitted white light without an optical filter.
Lane 1: 15ug, Lane 7: ~20ng, Lane 10: ~0.8 ng BSA.

Citations

View all 1 citations: Citation Explorer

Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis
Authors: Tomioka, Yui and Nakagawa, Masataka and Sakuma, Chiaki and Kurosawa, Yasunori and Nagatoishi, Satoru and Tsumoto, Kouhei and Arakawa, Tsutomu and Akuta, Teruo
Journal: Analytical Biochemistry (2022): 114817

References

View all 50 references: Citation Explorer

Ligand binding to a humanized anti-cocaine mAb measured by dye absorption spectroscopy.
Authors: Kirley, Terence L and Norman, Andrew B
Journal: Biochemical and biophysical research communications (2021): 93-98

Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA.
Authors: Leitner, Peter D and Vietor, Ilja and Huber, Lukas A and Valovka, Taras
Journal: Scientific reports (2021): 2331

Thermal Shift Assay for Exploring Interactions Between Fatty Acid-Binding Protein and Inhibitors.
Authors: Hao, Jiaqing
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 395-409

Thermal shift assay to probe melting of thrombin, fibrinogen, fibrin monomer, and fibrin: Gly-Pro-Arg-Pro induces a fibrin monomer-like state in fibrinogen.
Authors: Crossen, J and Diamond, S L
Journal: Biochimica et biophysica acta. General subjects (2021): 129805

SYPRO Orange - a new gold standard amyloid probe.
Authors: Mora, Aruna K and Nath, Sukhendu
Journal: Journal of materials chemistry. B (2020): 7894-7898

A novel differential scanning fluorimetry analysis of a humanized anti-cocaine mAb and its ligand binding characteristics.
Authors: Kirley, Terence L and Norman, Andrew B and Wetzel, Hanna N
Journal: Journal of immunological methods (2020): 112676

nanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability.
Authors: Real-Hohn, Antonio and Groznica, Martin and Löffler, Nadine and Blaas, Dieter and Kowalski, Heinrich
Journal: Frontiers in microbiology (2020): 1442

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes.
Authors: Rieser, Ruth and Penaud-Budloo, Magalie and Bouzelha, Mohammed and Rossi, Axel and Menzen, Tim and Biel, Martin and Büning, Hildegard and Ayuso, Eduard and Winter, Gerhard and Michalakis, Stylianos
Journal: Journal of pharmaceutical sciences (2020): 854-862

Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants.
Authors: Oshinbolu, Sheun and Shah, Rachana and Finka, Gary and Molloy, Mike and Uden, Mark and Bracewell, Daniel G
Journal: Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986) (2018): 909-917

Destructive twisting of neutral metalloproteases: the catalysis mechanism of the Dispase autolysis-inducing protein from Streptomyces mobaraensis DSM 40487.
Authors: Fiebig, David and Storka, Juliana and Roeder, Markus and Meyners, Christian and Schmelz, Stefan and Blankenfeldt, Wulf and Scrima, Andrea and Kolmar, Harald and Fuchsbauer, Hans-Lothar
Journal: The FEBS journal (2018): 4246-4264