Multiplex Western Blots

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

Azure fluorescence capabilities vs. competitor fluorescence, 3 proteins, plus total protein staining, and multiplex fluorescent 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*

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


– 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


– 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


– 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

Sapphire FL Biomolecular Imager

Imaging Solutions for Multiplex Fluorescent Western Blotting

With the Azure Sapphire FL 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: 5 – 1,000 microns

Dimensions (W x H x D): 75 x 45 x 70 cm

Weight: 63.5kg / 140lbs

Azure Sapphire FL Biomolecular Imager with lid open

The new Azure Sapphire FL Biomolecular Imager is capable of high resolution imaging and wide depth of field enable many sample types. Scan as many as 6 membranes (9 x 7 cm) or 6 96-well plates at a time. 4 cm of clearance above the glass allows for small animal imaging and tissue imaging.

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

NIR Fluorescence
Visible Fluorescence
Phosphor Imaging

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

Ready to multiplex like a pro?

Fill out this form to talk with an expert and get more information on the Azure Sapphire today!

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