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Analytical Chemistry and Chromatography Techniques

Applying Your MALDI Matrix Like a Boss

Matrix-assisted laser desorption/ionization (or simply, MALDI) is a type of mass spectrometry method. If you are new to mass spectrometry, check out our excellent primers, titled How Does Mass Spec Work and Get Out of Western Blot Hell: An intro to Mass Spec. Basically, however, mass spectrometry allows you to measure, sort, and quantify small…

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Examining Cell Interactions with Surface Plasmon Resonance (SPR) and Identifying Epitopes using SPR-Mass Spectrometry (MS)

Surface plasmon resonance (SPR) offers highly efficient, label-free detection for quantifying biomolecular interactions in real-time. Two exciting SPR variants that have sprung up in recent years are SPR for cellular analysis and SPR-mass spectrometry (SPR-MS). SPR for cellular analysis allows you to study how cells attach to different substrates and each other, while SPR-mass spectrometry…

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All Charged Up: The Basics of Ion-Exchange Chromatography

Highly pure proteins are vital for successful experiments; they play roles in research as assay reagents (especially for SPR applications), therapeutic candidates, and of course, as the subjects of structural and biochemical studies.  Chromatography is the science of separation and we utilize it to isolate and purify proteins based on their unique physiochemical properties. One…

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The Importance of Metabolomics Research

Metabolomics may sound like a fictional character in the famous comic series “Asterix”, but it is very important in understanding systems biology and in clinical research against various diseases. Metabolomics is the last piece of a puzzle of omics applications following genomics, transcriptomics, and proteomics. It allows us to ask “What has happened and what…

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Critical Factors for Full Characterization of Biosimilars

Today, about half of all therapeutic drug approvals by the US Food and Drug Administration (FDA) are for biological drugs. That number is expected to rise to 75 percent by 2025. When these biologics come off patent, the gates will open for a flood of biosimilar drugs (biopharmaceutical generics) that are designed to be much…

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Key Analytical Challenges for Antibody Drug Conjugates

Currently, there are more than 75 antibody drug conjugates (ADCs) in various stages of pre-clinical and clinical development. The combination of a targeted antibody coupled with a cytotoxic small-molecule drug (via  a flexible linker) makes for a lethal and specific oncologic drug product. However, an ADC is a heterogeneous cocktail of molecules with a range…

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Pack a Chromatography Column Like a Pro

You already learned the basics of column packing. When moving to more automated system using low pressure liquid chromatography systems, you can use pre-packed columns. But in order to compare several resins in specific conditions, and also to save money, you might need to pack your own low pressure columns. The art of packing a column…

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To Run or to Fly: A Comparison Between HPLC and GC

Many chemical compounds, including drugs and metabolites, can be analyzed by either gas chromatography (GC) or high-performance liquid chromatography (HPLC). Because both techniques function under the same basic principles of compound separation, identification, and quantification, it can sometimes be tricky to choose one over the other. But knowing what differentiates HPLC and GC can definitely…

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How to Clean and Unclog Your HPLC Column

In my last article, I discussed how to best keep your lab’s HPLC running smoothly. However, even the best-maintained HPLCs and columns need periodic cleaning. Today, I’ll describe how to identify and troubleshoot a clogged HPLC column. Columns Are Finite First of all, it’s important to realize that columns do have a finite lifetime. The…

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How to Separate Nucleotides Using Ion-paired Reverse Phase HPLC

If you work in the field of molecular biology, there is hardly a day that goes by that you don’t use nucleotides. But beyond the use of the four well-known deoxynucleotides in PCR, you can use nucleotides for several other applications. For example, kinases and phosphatases use nucleotides as substrates, and phosphotransferases transfer phosphate group…

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Using a Gel Filtration Chromatogram to Estimate Molecular Weight

Gel filtration chromatography (also known as size exclusion chromatography, molecular sieve chromatography, or gel permeation chromatography) is based on the differential distribution of the components in a sample between the mobile and stationary phases. Specifically, in gel filtration chromatography, this differential distribution depends on the size and shape of the components. Here, we take a…

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FTIR Spectroscopy: Every Window Needs a Good Acid Wash

Fourier Transform Infrared spectroscopy (FTIR spectroscopy) is a useful and exquisitely sensitive technique used to identify and quantify unknown compounds, as well as study fine molecular details. However, to obtain a meaningful IR spectrum, it is not only important to prepare the sample correctly but also to learn how to clean the apparatus that houses…

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Gel Filtration Chromatography Media: What You Need to Know

Do you use pre-packed Superdex 75 or Superdex 200 columns for gel filtration chromatography? Ever wonder if other media could better address your purification needs? Look no further. Your quest ends here! But if you’re looking for information on gel filtration principles, check out this earlier article. Choosing a Column for your Gel Filtration Experiment…

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How Separation Works in Column Chromatography Methods

Separation in column chromatography relies on differences. Molecules vary in size, charge, polarity, and solubility. We leverage these differences to distribute molecules between a stationary phase and a mobile phase. But because molecules are so different, it’s not possible to have a single method that works for all. In my previous article I discussed the…

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Fine-Tune Your MALDI-TOF to Produce Good-looking Mass Spectra

Mass Spec is all about getting the perfect peaks. Without a good peak assigning the correct mass is impossible and you cannot make accurate identifications. Make sure you know how to adjust your MALDI-TOF instrument parameters to achieve your perfect peak. In our previous posts ‘How does Mass Spec Work’ and ‘Imaging Mass Spectrometry: the…

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The Basics of Running a Chromatography Column

Column chromatography is a common technique used to separate individual compounds from a mixture. You can use column chromatography on both a small or large scale to isolate and purify material for use on a later experiment. If you are new to column chromatography or just want a refresher, then read on for a quick…

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Troubleshooting Thin Layer Chromatography: Some TLC for Your TLC

The whole TLC technique sounds easy to do, but it can be difficult and tricky during interpretation or give unexpected results, especially when working with biomolecules. For this reason, it is important to be familiar with troubleshooting thin layer chromatography. Some of the common problems faced during TLC and their solutions are listed below: Solvent…

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