![]() 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 about 10 minutes. Place the membrane on a piece of dry filter paper.Leave the membrane and filter paper on the benchtop for about an hour. Blots can be dried using any of the following options. Dry MembraneĪllow blots to dry before proceeding with detection. ![]() More information about transferring proteins to a membrane is available here /proteintransfer. Transfer to nitrocellulose or PVDF membrane. Run the gel according to the manufacturer's instructions. For example, you may need to use a different starting value for your dilution series to ensure that your target can be detected in every dilution. ![]() The actual amounts you load may vary depending on the particular conditions in your experiment. Using two 15-well gels, load the following samples in the order indicated in the table below. You will be loading a serial dilution in duplicate and in random sample order. 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.įollowing is a suggested template for loading a gel for the blocker optimization experiment. You can write on nitrocellulose membranes with pencil or the Odyssey Pen (PN 926-71804). Do not write on membranes with regular ink pens or markers, because the ink will fluoresce on Odyssey Imaging Systems.Be careful not to touch the membrane with your hands or gloves. Only handle membranes by the edges with clean forceps.Western blots should be prepared using standard blotting procedures and Millipore Immobilon-FL PVDF or Odyssey Nitrocellulose Membrane.Standard protein electrophoresis conditions and reagents can be used for gel and sample preparation. SDS (when using Immobilon-FL PVDF membrane) Methanol (when using Immobilon®-FL PVDF membrane) If you are using subclass specific antibodies, please refer to this technical note: Western Blot and In‑Cell Western™ Assay Detection Using IRDye Subclass Specific Antibodies ( /subclass). Primary antibodies must be from host species compatible with the secondary antibodies being used. Membrane: Odyssey Nitrocellulose (926-31090, 926-31092) or PVDF Membrane Kit with 4X Protein Loading Buffer (926-31097) Intercept (TBS) Protein-Free Blocking Buffer (927-80001)īlocking buffer of your choice (milk, BSA, etc.) Intercept (PBS) Blocking Buffer (927-70001) Intercept (TBS) Blocking Buffer (927-60001) Odyssey Protein Molecular Weight Marker (928-40000) If you use a PBS-based buffer system, choose Intercept® (PBS) Blocking Buffer. ![]() For example, if you use a TBS-based buffer system, choose Intercept® (TBS) Blocking Buffer. Be sure to keep your buffer system consistent throughout the protocol for blocking, antibody dilutions, and washes.Intercept® Blocking Buffer ( /intercept) is available in protein-based and protein-free formulations in TBS and PBS.TBS-based blocking buffers are generally used to detect phospho-proteins, because the phosphate present in PBS blocking buffers may competitively bind with antibodies to phospho-proteins.When choosing a blocking buffer, it is best to keep these considerations in mind: The experimental design suggested in this document is set up to let you compare three specific blocking buffers to a blocking buffer and buffer system of your choice.Īt this point, you can choose the blocking buffer that you would like to test. The buffering system will also be evaluated. The specific lysate and antibodies used in your system will be evaluated in four different blocking buffer solutions. This document describes a method to determine optimal blocking conditions for NIR Western blot detection with the Odyssey family of imagers. Blocking Buffer Optimization Protocol Introduction
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