Posts Tagged ‘Western Blotting’
What Ponceau S Staining Can Reveal About Your Western Blot
Discover how Ponceau S works, how to perform staining, and get a detailed guide to interpreting what the staining means when troubleshooting your failed blots.
Read More3 Hot Tips to Optimize Your Western Blot Transfers
Are you struggling with ugly and unreadable western blots? Here are 3 ways to optimize your western blot transfer and get blots to be proud of.
Read MoreHow SDS-PAGE Works
Knowing how SDS-PAGE works means that you can troubleshoot any issues in your experiment and tweak the setup to get publication-worthy figures. Find out how it works here.
Read MoreTop 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…
Read MoreHow 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…
Read MoreSDS-PAGE Tips and Tricks to Save You Time
Today, SDS-PAGE is one of the basic methods in biology labs. As a beginner in my lab, I had little help, so my first SDS-PAGE gel took two whole days! Since then, I’ve learned a lot about how to make the process faster. Here are some SDS-PAGE tips and tricks that I learned to help…
Read MoreHow to Cherry Pick Your Primary Antibody
How do you pick which antibody you should use in your assay? If you’re starting a new assay and need an antibody for the job, then selecting a new antibody from the plethora available could be high up on your to-do list.
Read MoreSonication – 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…
Read MoreCommon 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.
Read MoreBis-Tris Gels: Sharpen Up Your Protein Bands
A sprinkling of Bis-tris is all it takes improve your protein gels considerably. If you can afford it!
Read MoreWhere 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…
Read MoreThe Wet-chamber Method: How to use Less Antibody and Save Money
Want to save money on one of the most expensive steps of your Western blots? Then, use less antibody for significant cost savings!
Read MoreNon-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…
Read MoreStreamline Your Western Blots
Western Blotting is a long established method for which the protocol varies little from lab to lab. However, there are some new products that are available and some tweaks that can be made to the protocols that may improve your results and reduce the time it takes you to execute this popular technique. Save Time…
Read MoreHow 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…
Read MoreChoosing the Right Molecular Weight Marker for SDS-PAGE
When it comes to choosing a molecular weight marker to run on your SDS-PGE gels, there are a lot of options out there. How do you know which one is right for you? Read on for tips on what to consider when choosing a standard for your protein gels. Before you go about selecting a…
Read MoreHow 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…
Read MoreHow 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…
Read More3 Approaches to Western Blot Transfer
I think that transferring Western blots is one the most enjoyable tasks to do in a lab: it’s quick, it’s messy, and on some gleeful level, it feels like a child’s art project gone wrong. Of course, it’s also finicky and slippery and prone to tiny pitfalls that can noticeably affect the quality of your…
Read MoreHow 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…
Read MoreHow 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…
Read More5 Ways to Destroy Your Western Blot
Western blotting is a common lab technique used to detect and analyze proteins. It also happens to be a really long and complicated procedure, with many steps along the way that are easy to mess up. How do you make sure that your Western blot is successful? Avoid the following five ways to destroy your…
Read MoreSDS-PAGE: The Easy Way to Find the Wells
If you have ever attempted to load a SDS-PAGE gel only to miss the well, stab the divider, and then watch helplessly as your sample squirts off into the wrong well, then this tip is for you. The fortunate among us are able to use pre-cast gels with the wells outlined on the gel plate,…
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