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Western Blot Protocol

Western Blot Procedure Using EviFluor Quantum Dot Conjugates

This protocol is based on standard Polyacrylamide gel electrophoresis (PAGE) and Western blotting procedures. This procedure is a general guideline; some optimization may be necessary depending on the particular biological system.

Required Materials:

• Electroblotting Apparatus
• Power supply
• Rocking platform
• Washing and Incubation dish
• Gel Imaging instrument. (See recommended filter sets. Works in most gel imaging systems with a UV light source.)
• Blotting Paper
  • Nitrocellulose membrane (0.2 micron) – Recommended
  • PVDF (0.2 micron)
• Blocking Buffer
  • Starting Block (Pierce catalog #37542) – Recommended
• EviFluor Incubation Buffers
  • Starting Block or Sea Block (Pierce catalog #37527)
• Washing Buffers (diluted to 1X with DI water prior to use)
  • TBS 10x
  • TBST 10x

All membrane incubations are to be carried out in an appropriately sized container and performed on a rocking platform under continuous motion.

Procedure:

1. Wash membrane 3x for 4 minutes in washing buffer (TBS).
2. Incubate membrane in 10ml of blocking buffer for 1 hour at RT or, overnight at 4°C.
3. Incubate with primary antibody in binding buffer (TBS or starting block buffer) for 1 hour at RT. Prepare the primary antibody as recommended by supplier or use 1-2 ug/ml.
4. Wash membrane 3x for 5 min in TBST.
5. Incubate 1 hour at RT in incubation buffer with 1:1000 dilution of EviFluor conjugate.
   If performing your own conjugation, normalize concentration of your conjugate to the EviFluor concentration listed in the EviFluor Specification document.
6. Wash blot 3x for 4 minutes in (TBS).
7. Store moist at 4°C in starting block buffer until imaged.

Order EviFluor Quantum Dot Conjugates Below

Product Availability
Conjugate	Wavelength	Part Number	Concentration	Price
Goat anti-Mouse	520nm 600nm 620nm 680nm	EF-C11-GMO-0XXX	2.5uM	$289
Goat anti-Rabbit		EF-C11-GRA-0XXX		$289
Goat anti-Rat		EF-C11-GRT-0XXX		$289
Streptavidin		EF-C11-STR-0XXX		$439
Biotin		EF-C11-BT1-0XXX		$439
Specifications

Questions?