Materials Needed

  1. WesternDot™ kit (W10132, W10142) containing wash buffer (10X), blocking buffer, goat anti-rabbit or goat anti-mouse biotin secondary antibody, and Qdot® 625 streptavidin conjugate.

  2. Goat anti-rabbit or goat anti-mouse alkaline phosphatase secondary antibody or blocker solution containing secondary antibody from the WesternBreeze® chemiluminescence kit (WB7104, WB7106).

  3. Novex AP chemiluminescent substrate (WP2002) and substrate enhancer for nitrocellulose membranes (WP2003), or CDP-Star® substrate and enhancer from the WesternBreeze® chemiluminescence kit (WB7104, WB7106).

  4. Rabbit primary antibody against antigen 1 and mouse primary antibody against antigen 2.

  5. Orbital shaker or rocking platform.

  6. UV epi- or transilluminator/ethidium bromide filter/camera or fluorescence imaging system.

  7. Chemiluminescence imaging system or X-ray film.
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Ordering Information

Sku Name Size Price Qty
W10132 WesternDot® 625 Goat Anti-Mouse Western Blot Kit 1 kit USD 337.00
W10142 WesternDot® 625 Goat Anti-Rabbit Western Blot Kit 1 kit USD 337.00
WB7104 WesternBreeze® Chemiluminescent Kit, anti-mouse 1 kit USD 385.00
WB7106 WesternBreeze® Chemiluminescent Kit, anti-rabbit 1 kit USD 385.00

Immunodetection Protocol

Perform all steps on an orbital shaker or rocking platform rotating at 1 revolution/second.  Be sure that all solutions cover the membrane and move freely over and around it.  Note: the wash buffer can be from either the WesternDot™ or WesternBreeze® kits.  For optimal results, use the blocking buffer from the WesternDot™ kit.

  1. Transfer the proteins to a nitrocellulose or PVDF membrane and allow the membrane to dry.

  2. If you are using PVDF membranes, re-wet the membrane in 100% methanol for 30 seconds.

  3. Wash the membrane two times for 5 minutes each with 20 mL of ultra pure water to remove gel and transfer buffer components.

  4. Block the membrane for 1 hour in 10 mL of WesternDot™ blocking buffer.

  5. Incubate the membrane for 1 hour up to overnight (as recommended by the manufacturer) in the manufacturer’s recommended dilution of primary antibody in 10 mL of WesternDot™ blocking buffer.

  6. Wash the membrane three times for 5 minutes each with 20 mL 1X wash buffer.

  7. Co-incubate the membrane for 1 hour in a 1:2000 dilution of goat anti-rabbit or goat anti-mouse biotin secondary antibody and in a 1:2000 dilution of the alternate goat anti-rabbit or goat anti-mouse alkaline phosphatase secondary antibody in 10 mL of WesternDot™ blocking buffer.

  8. Wash the membrane three times for 5 minutes each with 20 mL 1X wash buffer.

  9. Incubate the membrane for 30 minutes to 1 hour in a 1:2000 dilution of Qdot® 625 streptavidin conjugate (0.5 nM) in 10 mL of WesternDot™ blocking buffer. 

  10. Wash the membrane three times for 5 minutes each with 20 mL 1X wash buffer.

  11. Wash the membrane one time for 5 minutes with 20 mL ultra pure water.

  12. Image the Qdot® 625 signal:   Qdot® 625 excites best with UV epi or transillumination.  Laser based scanners with an excitation source of 473-488 nm are also acceptable.  Qdot® 625 has an emission maximum at 625 nm, so emission is best captured using a 555 nm or 580 nm long-pass filter or band-pass filter that can capture 625 nm light.  With epi-illumination, exposure times vary with the intensity of the illumination source and concentration of the labeled proteins from 2 sec to 2 min.  Do not let the membrane dry during imaging, as that will reduce the activity of the AP enzyme for the subsequent chemiluminescent detection step.  Simply squirt a little water onto the surface of the imager and place the membrane face up over the water.  Wrapping the membrane in plastic wrap to reduce the drying rate will increase background fluorescence.  Multiplexed western detection can also be performed using PVDF membranes, but wet UV transillumination results in lower sensitivity and high background.  For best results with PVDF membranes, image with epi-UV illumination.

  13. If the same imager is to be used for imaging of the WesternBreeze™ AP chemiluminescent signal, then simply prepare the CDP-Star® substrate as directed and pipette approximately 2 mL of substrate over the surface of the membrane, completely covering the surface.  Try not to move the membrane from its position on the imager tray.  Allow the reaction to develop for 5 minutes and then expose for 1 minute to several minutes depending on the signal produced and the imaged desired.  If a longer development time or exposure is needed, then overlay a clean piece of plastic, such as copier transparency film, over the membrane to slow drying.  If the membrane has not been moved during acquisition of the two images, then the images can easily be overlayed without any manipulation of the orientation of the images.  If it is necessary to move the membrane to another imager or expose to X-ray film, then briefly return the membrane to the western staining dish and immerse in 20 mL water to fully rewet prior to incubating in the chemiluminescent substrate.

Note 1: The CDP-Star® chemiluminescent signal is not detected with UV excitation and emission filters used for Qdot® 625 imaging, so Qdot® 625 signal can be imaged during CDP-Star® substrate incubation or afterwards on wet or dried blots.

Note 2: WesternDot™ can be multiplexed with other chemiluminescent detection reagents, such as ECL (WP20005), or chromogenic detection reagents, such as WesternBreeze™ AP BCIP/NBT (WB7103, WB7105, WP20001).  These reagents can be detected in the Qdot® 625 image, so the Qdot® 625 signal must be imaged first, before addition of these detection reagents.

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