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Figure 1. AzureSpot Multiplex Image

Visualizing and Quantifying phosphoproteins via Western Blotting Part 2 of 2

Best practices for quantitation of unphosphorylated vs phosphorylated forms In part 1 we reviewed tips for using multiplexing to visualize phosphoproteins, and in part 2 we’re going to cover quantitation. Once you have reliable, repeated samples that fluoresce in non-overlapping bands and are linearly proportional to the signal, you can begin analysis. Here we will […]

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phosphoImg7

Visualizing and Quantifying phosphoproteins via Western Blotting Part 1 of 2

Western blotting is an important tool for researchers studying signal transduction and other processes where understanding the phosphorylation state of a protein is important for elucidating protein function. But some researchers are hesitant to use Western blotting to calculate the relative amount of phosphorylated to unphosphorylated protein. Here we discuss steps you can take to […]

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Western Blot Normalization

Quantitative Westerns: What is the best way to normalize your Western blot?

Far from being an “is-it-there-or-not” technique, modern digital detection instruments can make Western blotting reproducible and quantitative. By working within the linear dynamic range of your detection method and normalizing the data to control for variations in protein load and membrane transfer, you can get truly quantitative results. But what is the best way to […]

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Direct Indirect

The evolution of the ELISA and other immunoassays

If you’re reading this blog there’s a high chance that you use antibodies in your work, in some form of immunoassay. However, many of us rarely stop and think about where these antibodies come from and how their development has been fundamental for the evolution of life sciences. A brief history of the antibody Antibodies […]

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