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1. www.licor.com › bio › applicationsProtocol for Quantitative Western Blots - LI-COR Biosciences

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   Here are the basic steps of a quantitative Western blotting procedure. This workflow includes resources and references to help you get the best blots.

   • Troubleshooting

     If you encounter high background or unexpected bands, try a...

   • Resources

     Fluorescent Western Blot Detection Protocol; Western Blot...

   • Normalization Handbook

     Perform a Western blot with a serial dilution of sample (an...

   • Chemiluminescent Western Blots

     Determining the Linear Range for Quantitative Western Blot...

2. www.licor.com › bio › supportNear-Infrared Western Blot Detection Protocol

   www.licor.com › bio › support

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   • Introduction
   • Required Reagents
   • Quick Start Hints and Tips
   • Western Blot Detection Protocol
   • Guidelines For Multiplexed Detection
   • Adapting Western Blot Protocols For Odyssey Imagers

   The Western blot detection protocol covered in this document begins after the transfer step and continues to the imaging step. Hints are provided before the protocol to help you get started. Guidelines for Western blot detection and adapting your protocol for an OdysseyImager are provided after the protocol.

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   You can use TBS-based or PBS-based buffers with this protocol. Be sure to keep your buffer system consistent throughout the protocol for blocking, antibody dilutions, and washes. For example, if you use a TBS-based buffer system, choose Intercept® (TBS) Blocking Buffer. If you use a PBS-based buffer system, choose Intercept® (PBS) Blocking Buffer. 1. Bl...

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   Fuorescence detection with OdysseyImagers provides a quantitative detection method for Western blots. The following section includes basic tips to help you get started. The best transfer conditions, membrane, and blocking agent for experiments will vary, depending on the antigen and antibody.

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   For the best results, read the entire protocol carefully before beginning your experiments. 1. To learn more about optimizing your Western blots, read Good Westerns Gone Bad (licor.com/GWGBIR). 2. To learn more about gel transfer, read Protein Electrotransfer Methods and the Odyssey Infrared Imaging Systems (licor.com/proteintransfer).

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   Multiple antigens can be detected simultaneously on the same blot using IRDye® secondary antibodies and VRDye™secondary antibodies. When performing a blot, use the standard Western blot protocol with the following modifications: 1. Combine the primary antibodies in the antibody diluent. Incubate simultaneously with the membrane. The primary antibodi...

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   When adapting Western blotting protocols for Odyssey detection or using a new primary antibody, it is important to determine the optimal antibody concentrations. Optimization will help achieve maximum sensitivity and consistency, while minimizing background. Parameters such as primary antibody concentration, IRDye® secondary antibody or VRDye™secon...

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3. reedd.people.uic.edu › ReedLabWesternBlotLICOR Western Blot Protocol – Reed Lab

   reedd.people.uic.edu › ReedLabWesternBlot

   Nov 10, 2016 · LICOR Western Blot Protocol – Reed Lab. UIC College of Dentistry. Reagents: Matrix EXTRACTION BUFFER, per sample. 70 μl dH2O. 30 μl glycerol. 187.5 mM tris. 6% SDS. 100 mM DTT. PROTEASE INHIBITORS (HALT 100x; pn 78440) LYSIS BUFFER (NG2/CSP4) NP-40 (Life Technologies; FNN0021) Complete protease inhibitor (Roche, 11697498001)

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4. www.licor.com › bio › supportHousekeeping Protein Normalization Protocol - LI-COR Biosciences

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   For more information, see the Housekeeping Protein Validation Protocol (licor.com/HKP-Validation; LI-COR). This protocol describes how to use a housekeeping protein for Western blot normalization and quantitative analysis. This protocol is intended for use with near-infrared fluorescent Western blots. Keys for Success Keys for Success

5. People also ask

   How do you validate a two-color Western blot?

   • Antibody validation. Two-color Western blot detection requires careful selection of primary and secondary antibodies to prevent cross-reactivity. Always perform single-color control blots first to verify antibody specificity, and to identify possible interference from background bands.

   Housekeeping Protein Normalization Protocol - LI-COR Biosciences

   www.licor.com/bio/support/contents/applications/western-blots/housekeeping-protein-normalization-protocol.html

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   How do I perform a western blot?

   • When performing a blot, use the standard Western blot protocol with the following modifications: Combine the primary antibodies in the antibody diluent. Incubate simultaneously with the membrane. The primary antibodies must be from different host species or subclasses if the targets are close in molecular weight.

   Near-Infrared Western Blot Detection Protocol - LI-COR Biosciences

   www.licor.com/bio/support/contents/applications/western-blots/fluorescent-western-blot-detection-protocol.html?Highlight=on-cell

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   How do I verify a linear range in a western blot?

   • Verify Linear Range Perform a Western blot with a serial dilution of sample. The dilution series should include samples with concentrations above and below the concentration you expect to use. Quantify signal from the pan-specific antibody and modification-specific antibody. Export quantification data to a spreadsheet program.

   Normalization Handbook - LI-COR Biosciences

   www.licor.com/bio/support/contents/applications/western-blots/normalization-handbook.html

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