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Chemiluminescent Substrates

Chemiluminescent detection has established itself over the last two decades as a popular method for substrate detection as it provides excellent sensitivities, simple protocols and additional technique related benefits.

UptilightTM Chemiluminescent blotting substates

Chemiluminsecent
  • Leading brand Picogram & Femtogram detection ranges
  • Excellent Prices
  • 1 minute incubations
  • Long signal durations across the range

The Uptima range of UptilightTM chemiluminescent substrates go head-to-head with leading brands in terms of sensitivity and signal duration. They are optimized for detection of immobilized peroxidase, especially in Dot, Western, Southern & Northern blotting techniques on nitrocellulose, nylon and PVDF with signal registered on scanners or X ray film.

Cheshire Sciences provides these levels of detection at significant price savings. For the best results and to achieve maximum benefit from the price savings we recommend you always spend time optimizing your antibody concentrations. Starter pack sizes exist for all reagents at nominal cost.

UptilightTM High Sensitivity - < 1 Picogram of antigen

UptilightTM High Sensitivity supports picogram detection and displays higher signal / lower background than competitor products with a large dynamic range. Incubation times are just 60 seconds and reagent displays excellent signal duration.

 

High

Detection Range Working Vol. Product No. Description Instructions MSDS Price
Pico/Femto 50ml 98490E Uptilight™ High WB Chemiluminescent Substrate. 25ml of reagent A, 25ml of reagent B. Sufficient reagents for 450cm2.. pdf pdf £21.00
Pico/Femto 200ml 98490A Uptilight™ High WB Chemiluminescent Substrate. 100ml of reagent A, 100ml of reagent B. Sufficient reagents for 1,800cm2.. pdf pdf £73.00
Pico/Femto 500ml 98490B Uptilight™ High WB Chemiluminescent Substrate. 250ml of reagent A, 250ml of reagent B. Sufficient reagents for 4,500cm2.. pdf pdf £99.00
Pico/Femto 1 litre 98490C Uptilight™ High WB Chemiluminescent Substrate. 500ml of reagent A, 500ml of reagent B. Sufficient reagents for 9,000cm2.. pdf pdf £195.00

UptilightTM Ultra Sensitivity - Low femtogram of antigen

UptilightTM Ultra Sensitivity supports detection down to 1 femtogram and displays higher signal / lower background than competitor products with a larger dynamic range. Incubation times are just 60 seconds and reagent displays excellent signal duration.

 

Ultra

Detection Range Working Vol. Product No. Description Instructions MSDS Price
Femto 24ml 58372E Uptilight™ Ultra WB Chemiluminescent Substrate. 8ml of reagent A, 16ml of reagent B. Sufficient reagents for 216cm2.. pdf pdf £42.00
Femto 60ml 58372A Uptilight™ Ultra WB Chemiluminescent Substrate. 20ml of reagent A, 40ml of reagent B. Sufficient reagents for 540cm2.. pdf pdf £135.00
Femto 120ml 58372B Uptilight™ Ultra WB Chemiluminescent Substrate. 40ml of reagent A, 80ml of reagent B. Sufficient reagents for 1,080cm2.. pdf pdf £199.00
Femto 240ml 58372D Uptilight™ Ultra WB Chemiluminescent Substrate. 80ml of reagent A, 160ml of reagent B. Sufficient reagents for 2,160cm2.. pdf pdf £387.50
Independent Test Data

Uptilight High Sensitivity comparisons

Respective manufacturer protocols were followed in each instance. Lanes on blots are the same for each blot: 10, 5, 2, 1, 0.5, 0.2, 0 ng load per well, marker. Blots are mouse IgG on 12.5 % SDS-PAGE under reducing conditions..

Uptilight Ultra Sensitivity comparisons

Respective manufacturer protocols were followed in each instance.

The end-user did not have time to optimise antibody concentrations so there is extensive overstaining. Lanes

  • 1. Non-visible luminescent stnds
  • 2. 5ng load of protein of interest in protein mix
  • 3. 1ng load in protein mix
  • 4. 500pg load in protein mix
  • 5. 100pg load in protein mix
  • 6. 50pg load in protein mix
  • 7. - 11. As before but in extraction buffer rather than protein mix
  • 12. Nothing
  • 13. 100pg load in PBS
  • 14. 50pg load in PBS

Exposure

General Considerations

Uptilight High and Ultra sensitivity Chemiluminescent reagents

Overview.

Uptilight High Sensitivity

2 Reagents: Mix 1 part A & 1 part B
Stable at 4oC for 18 months
Sensitivity: <1 picogram

Uptilight Ultrasensitivity

2 Reagents: Mix 1 part A & 2 parts B
Stable at 4oC for 18 months
Sensitivity: Low femtogram

 

My blot is black/ reversed. Why?

It is likely you have too much HRP and your antibody concentrations are far too high. You need to dilute your antibodies far more.

What else can indicate too much HRP?

  • Inconsistent results
  • Signal dies very quickly
  • No signal at all (in this instance the signal dies so quick that it cannot be captured)
  • Brown on the membrane

What can too much HRP be due to?

  • Too high primary & secondary antibody concentrations
  • Using an inappropriate blocking reagent
  • Insufficient washing / lack of Tween 20 in the blocking & washing steps

Anything to avoid?

  • Milk! If you are using Avidin-Biotin systems, milk contains endogenous biotin.
  • Azide! Inhibits HRP (also sulphide, cyanide, fluoride & superoxide ions to some extent)