Multiplex Western Blots

Detecting one protein, two proteins, three proteins? How about FOUR proteins on the same Western Blot?

Save money on each experiment!

Fill out this form to learn how to save money with more quantitative results

Time and Cost Savings with Fluorescent Western Blotting*

ECL WBECL WBFLUORESCENCE WB
Detection MethodFilmCCD ImagerFluorescence Imager
# of Target Proteins2 Targets2 Targets2 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 hours8.3 Hours per WB7.4 Hours per WB4.3 hours per WB

*Western blot expenses based on the use of consumables. Calculations assume a 10x10cm Western Blot. Online prices, February 2021.

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

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

Azure Sapphire Biomolecular Imager

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

Product Specifications:
Up to 4 solid-state diode lasers excitation sources (40,000+ hour lifetime)
Up to 5 high quantum efficiency emission detection channels
Fluorescent Detection Method: APD + PMT​
Visible Fluorescence
Phosphor Imaging
Chemiluminescence with CCD camera
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 x 25 cm
Resolution: 10 – 1,000 microns
Dimensions (W x H x D): 75 x 45 x 70 cm
Weight: 63.5kg / 140lbs

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.

The Ultimate Fluorescent Scanning System

AZURE SAPPHIRE ™LI-COR ODYSSEY® CLX®AMERSHAM® TYPHOON®
NIR Fluorescence
Visible Fluorescence
Chemiluminescence
Phosphor Imaging
Densitometry
Resolution10µm21µm10µm
Price$$$$

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
Publications

Ready to multiplex like a pro?

Connect with us to get more information on the Azure Sapphire today!

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