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Protein Expression and Analysis

Let me introduce you to ELISA…No, not the girl…The assay.

An ELISA (Enzyme-Linked ImmunoSorbant Assay) is a popular assay that uses antibodies and color change to detect proteins, peptides, antibodies or biomolecules in complex mixtures. ELISAs are popular because they are reliable, specific, easy to use, and can easily be scaled up to process multiple samples simultaneously. How an ELISA is Done: In an ELISA,…

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Focus on Isoelectric Focusing

Isoelectric focusing electrophoresis (IEF) of proteins is nowhere near as popular as its cousin – sodium dodecyl sulphate-polyacrylamide gel electrophoresis aka SDS-PAGE. While in both methods the proteins are denatured, IEF is a gel-based electrophoretic separation of proteins using difference in their overall charges. The sodium dodecyl sulphate – SDS part of the usual gel…

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How to Identify Protein Motifs from Protein Sequences

Wouldn’t it be great to put your nucleotide sequence into a program and get back a 3D-structure of your protein and a full description of its functions? In theory, because the protein 3D-structure is determined by the aminoacid sequence, given the right algorithm and a powerful enough computer, this should be simple.  In practice, because…

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Counting On Your Results – Tips And Technologies For Colony/Plaque Counting

Have you ever emerged from the lab, bleary-eyed, blinking dazedly at the sun after spending hours hunched over a lab bench counting endless bacterial colonies or viral plaques? A necessary evil… I consider colony/plaque counting one of the necessary evils of working with microorganisms.  Necessary because many experiments have an endpoint that requires determining the…

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Breaking Up is (Not That) Hard to Do: Sonication for Cell Lysis

To answer some of the more interesting research questions, you often need to get a good look at what’s going on inside the cell. Whether you’re running a Western blot or measuring enzyme activity, many assays require access to the materials (e.g. proteins, DNA, subcellular fragments) contained within the cell walls. There are several ways…

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Understand EC numbers in 5 minutes Part 2: History of the EC system

In the previous article on EC numbers, I explained how the Enzyme Commission names enzymes, and why it is so important. In this article I’d like to take you on a brief journey through the history of the Enzyme Commission. Like many histories in science (e.g. this!), it is fascinating and gives a useful perspective…

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Understand EC numbers in 5 minutes Part I: How EC numbers work

As a biologist you will no doubt have seen Enzyme Commission (EC) numbers. An EC number is group of four numbers separated by periods in papers discussing enzymes…something like this: EC 1.1.2.1. But do you know what these numbers mean? Or where they came from? Or why we use them? If not, I will aim…

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Tips For Better ELISA Results

The ELISA (enzyme-linked immunosorbent assay) is a rapid method used to detect the amount of a protein of interest in clinical and experimental samples. There are a number of ELISA formats to choose from, depending on your research needs. These include direct ELISA, indirect ELISA, competitive ELISA and sandwich ELISA. We have previously covered the…

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Second Chance Saloon: How to Western Blot a Coomassie-stained gel

In her article How to Get Perfect Protein Transfer in Western Blotting, Emily Crow recommends Coomassie staining your gel after transfer to the membrane to check the quality of the transfer. A good transfer should not leave behind proteins and PVDF membrane, stained by 0.1% Ponceau S in  5% phosphoric acid and destained with water…

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Tips for Choosing and Using a New Primary Antibody

Finding a good primary antibody can often feel like playing Russian roulette.  Nothing is more disappointing than buying a $300 antibody that doesn’t work for your use. There are some steps you can take, however, to increase your likelihood of success. Scout out Other Labs Before you buy, ask if anyone around you or in…

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How Does 2D Gel Electrophoresis Work?

2D gel electrophoresis (2DE) is a key technique for purifying individual proteins from complex samples based on their isoelectric points and molecular weights.  Simple enough in theory, but as the plethora of protocols and articles shows, 2DE demands patience and meticulous optimization.  But whether your samples are human sera or HUVEC lysates, 2DE uses these…

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Kits And Reagents For Mammalian Cell Transfection

Transfection of animal cells has proven an invaluable tool in studies of gene expression, cell behavior, cell processes and molecular genetics. Essentially, transient pores are opened within the cell’s lipid bilayer, allowing insertion of nucleic acids (DNA, RNA, siRNA, RNAi), proteins, nanoparticles, and even antibodies into the cellular milieu. Transfection can be achieved using many…

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Top Ten Tips for TAP

Tandem affinity purification (TAP) is a versatile technique allowing the isolation of proteins for various purposes including Western blot and mass-spectrometry. The target protein is fused with protein A from streptococcus and the calmodulin binding domain, which together comprise the TAP-tag (for an introduction to TAP-tagging, see this article). To purify a TAP-tagged protein from…

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Five Things You Might Not Know about LB

LB medium is a staple in virtually every lab. It’s commonly used to propagate E. coli, and as such will be used frequently by any lab that does cloning. Chances are, LB broth or plates were one of the first things you learned to make as a newbie in the lab.  Here are a few…

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How to Reduce Antibody Contamination When Western Blotting Co-IPs

Co-immunoprecipitation is a method used to detect protein-protein interactions. While it can be wonderful when it works, there are many problems associated with this technique. One of the biggest problems that I have faced when using this method is contamination by the light and heavy chains of my precipitating antibody when performing western blots of…

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Spotter’s Guide to Protein Tags

Protein tags are invaluable tools for the modern biologist, particularly if you work on one of the 99% of proteins for which there isn’t a nice antibody readily available. If you want to purify large amounts of your protein of interest, detect it by western blot or fluorescence microscopy, or identify its potential binding partners,…

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How Do YOU Image Your Western Blots?

The last step in western blotting is imaging the blot – this is the moment of truth, when you finally get to see the results of the experiment you’ve been working on for so long!  There are a variety of different ways to image your blot.  The method you choose will largely depend on the…

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How to Lyse Cells for Protein Extraction

The first step in most Western blotting experiments is lysing your cells to extract protein.  You need to break open your cells in order to be able to isolate the proteins, and you need to do this with the least degradation and the most reproducibility possible.  Depending on what your starting material is, there are…

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How Proteases and Protease Inhibitors Work

Proteases: wild, mysterious, destructive.  What are these untamed elements ravaging your precious lysate? How can a drop of EDTA or a smidge of “cocktail” protect that sample, which is gently cradling your hopes, your dreams, and your desire to survive the next lab meeting? Brace yourself for a biochem flashback: in this article, we’ll explain…

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Wasting Antibodies Doesn’t Float Your Boat? Try Floating Your Blot Instead!

Western blots may be great for visualizing protein expression, but they can be a perfect way to waste your precious antibody stocks if you follow the normal protocol. Thankfully, you don’t have to follow the normal protocol any more; here’s how to get great blots with a fraction of the antibody usage. I have tried…

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Film vs. Digital: A Real Western (Blotting) Showdown

You’ve masterfully run and transferred your gel, and now it’s time to probe and quantify your protein(s). You’ve got your antibodies and ECL ready to go. Substrate – check. Film cartridge – check. Darkroom – ah yes, that magical place where night vision goggles are required to navigate a veritable minefield of potential chaos. It…

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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…

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Choosing 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…

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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…

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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…

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3 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…

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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…

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An Introduction to Tandem Affinity Purification

Tandem affinity purification is a development of existing techniques for purifying protein complexes from cells in physiological conditions. It was first described over ten years ago and has become a commonplace laboratory tool. In this brief article I’ll introduce the basic technique and describe some of its advantages. Biology is a team game. Most biological…

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When Silence Speaks Volumes: Using RNAi to Investigate Gene Function

RNA interference (RNAi) may have originated as a defense mechanism to protect cells against foreign genes introduced by viruses. This concept has since been put to use to create a powerful experimental tool for investigating gene function in organisms. Small-interfering RNA (siRNA) libraries for investigating genome-wide function can be produced by chemical synthesis of probes…

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5 Ways to Improve Your ELISAs

ELISAs (enzyme-linked immunosorbent assays) are often used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones in research and diagnostics. Today, a wide range of ELISA formats exist to suit your needs e.g. indirect ELISA, direct ELISA, competitive ELISA and sandwich ELISA. While it has become easier to perform ELISAs, thanks to…

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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…

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Detecting Protein Phosphorylation Without Radiation Phospho Specific Antibodies Or Mass Spec

Phosphorylation is one of the major post-translational modifications that regulate the activity of a protein. Around a third of human proteins are believed to be phosphorylated, and so the kinases and phosphatases that mediate protein phosphorylation are of major interest to biomedical researchers. However detecting protein phosphorylation can be difficult, particularly from cell extracts. Phospho-specific…

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Six Fixes For Antibody Co-Elution In Immunoprecipitations

Do you want to immunoprecipitate (IP) a protein with a molecular weight that is anywhere near 55 kDa or 25 kDa? Then you have an irritating problem to deal with: antibody co-elution. But don’t panic, we have six strategies for dealing with your new problem. The Problem: Typically, the IP antibody is bound to Protein…

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Should You Use Magnetic Beads for Immunoprecipitation?

Sepharose beads are porous, which gives them a high surface area for interaction with proteins and allows them to hold a lot of liquid. This is perfect for the application that they were originally designed for: purifying milligrams of protein in columns. When immunoprecipitation (IP) – a small-scale technique for pulling specific proteins out of solution using…

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Working with Enzymes: Part I -The Simple Kinetic Spectrophotometric Assay

At the end of my last article, I provided some practical tips and tricks for working with enzymes at the bench. Now, we’ll cover one of the cornerstone techniques of enzymology work: the enzyme assay. Starting with the simple assays and eventually working our way to the more complex, this article introduces the principles of…

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5 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…

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Working with Enzymes: Stability, Purification and Activity

In Part 1 of this series, we began our journey into the fascinating world of enzymology. We looked at the most basic concepts of what an enzyme is and the incredible jobs it can do. In Part 2 of “Working with Enzymes,” I will look at some things that you should keep in mind to…

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The Ins and Outs of Protein Concentration – Chromatography

In parts one and two of this series I described how semi-permeable membranes and precipitation methods could be used to concentrate your protein-of-interest, but there is one more method that you may not have thought of for protein concentration – chromatography. While chromatography resins are an obvious choice for protein purification, they can also be…

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The Ins and Outs of Protein Concentration – Protein Precipitation

While precipitation is an obvious choice for concentrating DNA and RNA samples, it can also be an effective way to concentrate proteins. Here in installment two of this three part series, I describe the two most common methods for protein precipitation – ammonium sulfate and trichloroacetic acid. Background Precipitation of proteins occurs primarily by hydrophobic…

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The Ins and Outs of Protein Concentration – Semi-permeable Membranes

This is the first of a three part series describing some of the most common methods for concentrating proteins. In later installments I’ll discuss using protein precipitation and chromatography to concentrate a protein. However, here I’ll detail the most popular approach – semi-permeable membranes, used for both dialysis and commercial protein concentrators. Structure of the…

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