VisULite MAX ECL Western Blotting Substrate
R&D Systems, part of Bio-Techne | Catalog # VL002
Key Product Details
Summary for VisULite MAX ECL Western Blotting Substrate
Kit Summary
Ready-to-use ultra high sensitivity Enhanced Chemiluminescent (ECL) substrate.
Key Benefits
- High sensitivity – femtogram level detection
- High signal intensity
- Excellent signal to noise ratio
- Compatible with X-ray film or CCD imaging systems
- Substrate A, 100 mL
- Substrate B, 100 mL
Stability and Storage
Store in the dark at 2 °C to 8 °C. DO NOT FREEZE.
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| Detection of GAPDH in HeLa Cell Llysates Visualized with VisULite MAX™ ECL Western Blotting Substrate Using X-ray Film or CCD Imaging System. A two-fold dilution series of HeLa whole cell lysate was prepared and loaded at 625, 313, 156, 78, 39, 19, 10 and 5 ng per lane. Samples were transferred onto PVDF membrane and probed with 0.1 µ/mL of Mouse Anti-Human/Mouse/Rat GAPDH Monoclonal Antibody (R&D Systems®, Catalog # MAB5718) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (R&D Systems®, Catalog # HAF018). VisULite MAXTM ECL substrate was applied for 1 minute, followed by exposure to a standard sensitivity film (left) or image capture using ProteinSimple® FluorChem™ M (right). Lanes are visualized on a 30 second exposure (film) or 1 minute exposure (imager). |
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| Detection of Recombinant Human CRP Visualized with VisULite MAX™ ECL Western Blotting Substrate. A two-fold dilution series was prepared using rhCRP (R& Systems®, Catalog # 1707-CR) and loaded at 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 pg per lane. Samples were transferred onto PVDF membrane and probed with 0.5 μg/mL of Sheep Anti-Human CRP (R& Systems®, Catalog # AF1707) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (R& Systems®, Catalog # HAF016). VisULite MAX™ ECL substrate was applied for 1 minute, followed by exposure to a standard sensitivity film for 15 seconds. |
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| Signal Duration with VisULite MAX™ ECL Western Blotting Substrate. A two-fold dilution series of HeLa whole cell lysate was prepared, loaded, transferred and probed with Mouse Anti-Human/Mouse/Rat GAPDH Monoclonal Antibody (R& Systems®, Catalog # MAB5718). VisULite MAX™ ECL substrate was applied for 1 minute to the PVDF membrane, followed by exposure to a standard sensitivity film for 1 minute at the indicated timepoints from 1 to 60 minutes after VisULite MAX™ ECL substrate addition. |
Assay Procedure
Refer to the product datasheet for complete product details.
- See MSDS
- 100 mL VisULite MAX™ ECL Western Blotting Substrate A
- 100 mL VisULite MAX™ ECL Western Blotting Substrate B
Allow VisULite MAX™ ECL Western Blotting Substrate A and Substrate B to equilibrate to room temperature approximately 30 minutes before use. Mix equal volumes of VisULite MAX™ ECL Western Blotting Substrate A and Substrate B. Prepare only the amount of VisULite MAX™ ECL Western Blotting Substrate needed for your experiment.
Run SDS-PAGE gel and transfer to nitrocellulose or PVDF by established laboratory protocol.
Label and block the membrane.
Incubate the membrane with primary antibody. Note: High sensitivity of substrate may require adjusting established parameters for primary and secondary antibody.
Wash and incubate with appropriate HRP-labeled secondary antibody.
Wash and for best results, use 0.2 M Sodium Phosphate for the final wash. At the completion of the wash, drain off all liquid and gently blot dry on a lab wipe or filter paper. Note: Do not let membrane dry completely.
Place membrane on clean plastic wrap and add approximately 100 µL of VisULite™ ECL Western Blotting Substrate per square centimeter of membrane. Allow to incubate for 1 minute.
Drain excess substrate and gently blot dry as above.
Place membrane in CCD camera system and expose according to manufacturer’s instructions or place membrane on clean plastic wrap (or equivalent) and expose to film.
Adjust exposure times as needed.
- FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
- This reagent should not be used beyond the expiration date indicated on the label.
- Results may vary due to variations in blotting conditions.
- Note: Because VisULite™ ECL Western Blotting Substrate is a highly sensitive substrate, optimization of primary and secondary antibodies may be required to generate consistently reproducible results with strong signals and low backgrounds. See www.rndsystems.com/resources/protocols/western-blot-cell-lysate-protocol for additional optimization tips and hints that may further improve performance.
- Do not allow membrane to completely dry during procedure.
- Do not use Sodium Azide in any buffers used for detection. Azide is an inhibitor of HRP.
- Work quickly once the membrane has been exposed to VisULite™ ECL Western Blotting Substrate to capture maximum signal.