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. Signal was detected with WesternBright® ECL substrate.Blocking Buffer Optimization Protocol Introduction The membranes were blocked with AdvanBlock™- Chemi before incubation with a rabbit anti-human STAT-1 (Millipore #06-501) primary antibody. An intact membrane was processed side-by-side a membrane that was partitioned and incubated in three chambers of a multi-chamber incubation tray. HeLa cell lysate was separated by SDS-PAGE and transferred to a PVDF membrane. Western blot membranes processed in Multi-chamber incubation trays are comparable to standard incubation trays. Each Multi-chamber incubation tray is partitioned into six chambers that measure 2.7 x 8.1 cm. These specially designed incubation trays are ideal for staining and washing electrophoresis gels and membranes that have been partitioned. Opaque containers are ideal for light-sensitive applications transparent containers are ideal for easy monitoring of colorimetric staining traditional containers are convenient for typical blot washes and incubations. The three available designs are all available in a range of sizes.
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