Do not add SDS if using a nitrocellulose membrane.If you are using PVDF, add 0.01 – 0.02% SDS to the diluted secondary antibody.Use Intercept T20 Antibody Diluent or blocking buffer with 0.2% Tween® 20 to the primary and secondary antibody solution.Avoid adding detergent during the blocking step.Background can result from membrane autofluorescence, blocking buffer, or non-specific binding of antibodies. Be careful not to touch the membrane with your hands or gloves.Ī low-background membrane is essential for fluorescent Western blot success. Only handle membranes by the edges with clean forceps. Use only a pencil to write on PVDF membranes, because the ink from the Odyssey Pen will dissolve in the methanol used to wet the PVDF membrane. You can write on nitrocellulose membranes with pencil or the Odyssey Pen (PN 926-71804). Incubate membranes with secondary antibodies in the dark for 1 hour with gentle shaking.ĭo not write on membranes with regular ink pens or markers, because the ink will fluoresce on Odyssey Imaging Systems. If particulates are seen in the antibody solution, centrifuge before use. Minimize exposure to light and take care not to introduce contamination into the vial.ĭilute immediately prior to use. Store the IRDye® secondary antibody or VRDye™ secondary antibody vial in darkness at 4 ☌. To use Revert™ 520 Total Protein Stain or Revert™ 700 Total Protein Stain for normalization, rewet the membrane (see Step 1) and see its pack insert for more information. A drawer or a cabinet shelf may suffice.įor weak or low abundance targets, 800 nm channel detection is recommended for best results. Place the membrane between two pieces of dry filter paper, and place the membrane and filter papers into a protected place overnight.Leave the membrane and filter paper in a 37 ☌ oven for approximately 10 minutes. Place the membrane on a piece of dry filter paper.Leave the membrane and filter paper on the benchtop for approximately 1 hour. They can be dried using any of the following options. Let the membrane dry after transfer to maximize protein retention on the membrane. The best transfer conditions, membrane, and blocking agent for experiments will vary, depending on the antigen and antibody. The following section includes basic tips to help you get started. The Odyssey M Imager is the only Odyssey Imager that can detect VRDye secondary antibodies.įuorescence detection with Odyssey Imagers provides a quantitative detection method for Western blots. IRDye® 800CW, 680RD, or 680LT secondary antibodies or VRDye™ 490 Secondary Antibodies or VRDye™ 549 Secondary Antibodies Intercept Blocking Buffer (PBS or TBS), Intercept Protein-Free Blocking Buffer (PBS or TBS) If you use a PBS-based buffer system, choose Intercept® (PBS) Blocking Buffer.īlotted nitrocellulose ( LI‑COR, PN 926-31090 or 926-31092) or Immobilon®-FL PVDF membrane ( LI‑COR, PN 926-31099 or 926-31100) For example, if you use a TBS-based buffer system, choose Intercept® (TBS) Blocking Buffer. You can use TBS-based or PBS-based buffers with this protocol.īe sure to keep your buffer system consistent throughout the protocol for blocking, antibody dilutions, and washes. Guidelines for Western blot detection and adapting your protocol for an Odyssey Imager are provided after the protocol. Hints are provided before the protocol to help you get started. The Western blot detection protocol covered in this document begins after the transfer step and continues to the imaging step. Near-Infrared Western Blot Detection Protocol Introduction
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