Top Five Methods for Primary Antibody Labeling

Top Five Methods for Primary Antibody Labeling

In any application that uses antibodies for signal detection (e.g., Western blotting, ELISA, immunohistochemistry, or FACS), there are two approaches to antibody labeling: direct and indirect labeling. Standard Western blotting uses indirect labeling because you use a primary antibody to detect the target antigen, followed by a secondary antibody to which a detection molecule is…

How to Choose Quality Antibodies for Successful Western Blotting
|

How to Choose Quality Antibodies for Successful Western Blotting

Successful western blotting means achieving unambiguous results, and this requires a sensitive and specific antibody-antigen interaction. Consequently, high quality antibodies are critical for reliable and consistent western blotting. Western Blotting Process In the basic western blotting process, polyacrylamide gel electrophoresis (PAGE) separates a mix of proteins according to their molecular weights (denaturing gels) or their…

Sonication – 7 Tips for Mastering the Art

Sonication – 7 Tips for Mastering the Art

Sonication is mostly used during preparation of protein extracts to help break apart the cell. Although most lysis buffers have buckets of detergent that lyse cell membranes, sonication just gives an extra hand in breaking everything apart. Sonication also breaks up, or shears, DNA in a sample—preventing if from interfering with further sample preparation. Have…

Common Mass Spectrometry Contaminants: I Messed It Up So You Don’t Have To!

Common Mass Spectrometry Contaminants: I Messed It Up So You Don’t Have To!

Through many trials, and lots of error, I learned that there are many considerations for mass spectrometry that might not be obvious to you as a molecular biologist. Common contaminants, even in small quantities, can mask important peaks in your mass spec data and have a huge impact on the final results.

Where are My Bands? Troubleshooting a Signal-less Western

Where are My Bands? Troubleshooting a Signal-less Western

Western blotting uses electrophoresis and antibody-epitope affinity to give a semi-quantitative and (theoretically) clear measure of protein abundance. It’s a long procedure, filled with many steps—and even more room for error. Learning to troubleshoot certain problems is incredibly important for continued success with this technique. So what do you do when your final imaged product…

Non-specific Binding? Tips to Sharpen up Your Western Blot

Non-specific Binding? Tips to Sharpen up Your Western Blot

In the previous installment of this series on western blotting, we addressed potential sources of error when your final product is completely bare. But alternatively, what do you do when too much background is the problem? You may have beautiful bands of interest—but if there is a bunch of non-specific binding, your quantification and data…

How To Preserve Your Samples In Western Blotting

How To Preserve Your Samples In Western Blotting

When running a quantitative Western blot, it’s crucial that your sample preparation is consistent.  Incomplete protein extraction from one sample will skew your results when you compare it to the protein content of a sample that was extracted more thoroughly.  And after the protein extraction, it’s important to handle the samples in an identical manner…

How To Preserve Your Samples In Western Blotting

How To Preserve Your Samples In Western Blotting

When running a quantitative Western blot, it’s crucial that your sample preparation is consistent.  Incomplete protein extraction from one sample will skew your results when you compare it to the protein content of a sample that was extracted more thoroughly.  And after the protein extraction, it’s important to handle the samples in an identical manner…

How to Optimize Your Western Blot Transfers

How to Optimize Your Western Blot Transfers

So, you’ve done your experiment, prepped your samples, and run your SDS-PAGE gel.  Now it’s time for the all-important transfer step, that tricky point that will determine the quality of your Western blot. Transfer times are empirical and based on your own particular samples, which means that there is no easy way to determine how…

How Do YOU Make Sure That Your Western Blots are Evenly Loaded?

How Do YOU Make Sure That Your Western Blots are Evenly Loaded?

For Western blot data to be reliable, it is important that you load known amounts of sample into each lane of the gel.  This is of particular importance if you are doing a quantitative blot, where you really need to be able to compare band intensity in each sample.  In this article, we’ll talk about…

How to transfer one SDS-PAGE gel onto two membranes

How to transfer one SDS-PAGE gel onto two membranes

Have you ever wished you could transfer the same SDS-PAGE gel twice? Sometimes, when you are blotting for many different proteins of similar size, stripping and reprobing multiple times can become impractical.  Here’s a simple diffusion transfer method that can be used to generate duplicate membranes from a single gel: Take a glass plate, or…