Multiplex Western Blots
Detecting one protein, two proteins, three proteins?
How about FOUR proteins on the same Western Blot! Save money on each experiment!
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Advantages to Fluorescent Multiplex Western Blots with the Azure Sapphire
|THE REACTION||- Easily adapt any ECL WB to fluorescence and save money in every experiment|
|- Stable fluorescent signal: no rush to image your WBs, membranes can be scanned after years|
|- Broadest range of fluorophores: Blue, Green, Red, NIR 700, NIR 800-|
|- Detection of up to 4 multiplexed reactions per WB|
|THE SCANNER||- 4 fluorescence channels to easily quantify overlapping bands|
|- Multiple channels to simplify normalization: no stripping and re-probing required|
|- Simultaneous multichannel scanning to reduce your imaging time|
|- Highest scanning speed|
|THE DATA||- Widest Dynamic Range to quantify extremely low-and high-abundance targets|
|- Greatest Linearity: direct proportionality of the fluorescent signal|
|- Highest resolution up to 10 microns|
Time and Cost Savings with Fluorescent Western Blotting
|Experiment Type||ECL WB||ECL WB||Fluorescence WB|
|Detection Method||Film||CCD Imager||Fluorescence Imager|
|# of Target Proteins||2 Targets||2 Targets||2 Targets|
|Protein Markers (0.5-1uL for IR; 10uL for Chemi)||$2.84||$2.84||$0.28|
|Secondary Antibody (1:10,000 for FL ; 1:20,000 for ECL)||$0.20||$0.20||$0.68|
|ECL Substrate (0.1 ml/cm2)||$32.80||$32.80||$0.00|
|Photographic Film (2-4 sheets/membrane)||$9.96||$0.00||$0.00|
|Stripping Buffer (20 ml/reaction)||$5.06||$5.06||$0.00|
|Total Experiment Cost||$50.86||$40.90||$0.96|
|Labor hours||8.3 Hours per WB||7.4 Hours per WB||4.3 hours per WB|
Western Blot expenses based on the use of consumables. Calculations assume a 10x10cm Western Blot. Online prices, February 2021.
Reliable Western Blot Normalization Using Fluorescent Total Protein Staining
Quantitative Western Blots require the normalization of each target’s signal to an internal loading control. Guidelines from leading journals state that total protein normalization is preferred over normalization to housekeeping proteins (HKPs).
- Use the Azure Sapphire scanner to normalize up to 3 target proteins against a HKP.
- Solve the lack of linearity of HKPs at high protein loads. Take advantage of the broad linear range of Azure TotalStain Q to normalize up to two targets in NIR WBs when lowly expressed proteins require high loads of protein to reach the required sensitivity.
Imaging Solutions for Multiplex Fluorescent Western Blotting
With the Azure Sapphire Biomolecular Imager, reimagine what your digital blot imager can accomplish.
Fluorescence | Chemiluminescence | Phosphor Imaging | Densitometry
- Up to 4 solid-state diode lasers excitation sources (40,000+ hour lifetime)
- Up to 5 high quantum efficiency emission detection channels
- Optional Phosphor Imaging Module
- Optional Chemiluminescence Module: CCD Camera
- Scanning Resolution: 10 – 1,000 microns
- Dynamic Range: 5 orders of magnitude, 16 bits (2^16= 65,536 levels between maximum and minimum signal)
- Max. scanning speed: 50 cm/s
- Max. Scanning area: 25 cm x 25 cm
- Dimensions (W x H x D): 75cm x 45cm x 70cm. Weight: 63.5 kg / 140 lbs
Azure Sapphire Biomolecular Imager
The Ultimate Fluorescent Scanning System
|Azure Sapphire ™||LI-COR Odyssey® CLx®||Amersham® Typhoon®|
|Resolution||10 μm||21 μm||10 μm|
Quantitative Confidence with Fluorescent Western Blotting
Quantitative western blots require the normalization of signal to an internal loading control. Guidelines from leading journals state that total protein normalization is preferred over normalization to housekeeping proteins.
- Utilize up to three channels for proteins of interest and the fourth for an internal loading control
- Experience no crosstalk between fluorescent channels
- Easily identify proteins with small shifts in molecular weight (ie. post-translational modifications), without stripping and reprobing
Journal of Proteomics
Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots
“Total protein staining (TPS) represents the actual loading amount more accurately than HKPs due to minor technical and biological variation. Further, the broad dynamic range of TPS solves the issue of HKPs that commonly fail to show loading differences above small loading amounts of 0.5-10 μg.”
Moritz CP. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics. 2017 Oct;17(20). doi: 10.1002/pmic.201600189. PMID: 28941183
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