Mentors at Your Benchside – Details, episodes & analysis

#112 — Isoelectric focusing (IEF) is a powerful technique distinct from the more familiar SDS-PAGE, [1,2] tailored for separating proteins or peptides based on their isoelectric points (pI). [3]

This method capitalizes on the migration of charged molecules through a stable pH gradient until they reach a zone where their net charge is zero, halting their movement.

Essential to setting up an IEF experiment are the IPG strips, equipped with a pH-responsive gel, and the sample, often mixed with carrier ampholytes for efficient migration.

IEF's utility spans various applications, from enhancing 2D-PAGE protein separations to analyzing post-translational modifications and preparing samples for mass spectrometry. [4]

Discover more about how this technique can advance your research and streamline your experimental workflows.

Resources:
1. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/
2. Isoelectric Focusing: A Simple Way to Enhance Your Protein Separation. Available at: https://bitesizebio.com/26811/isoelectric-focusing-separation-proteins-peptides/
3. The Definitive Guide to pH, pKa, and pI. Available at: https://bitesizebio.com/9425/what-is-ph-pka-pi/
4. Detecting Post-Translational Modifications. Available at: https://bitesizebio.com/49322/detecting-post-translational-modifications/

#111 — In this episode, we dive deep into the fascinating world of microbes and their revolutionary applications in biotechnology. From environmental solutions to breakthroughs in health and medicine, microbes hold the key to some of the most advanced scientific developments.

Discover how these microscopic organisms are transforming industries and pushing the boundaries of what's possible in science and technology. We’ll explore the latest research and applications that are shaping the future.

For an in-depth look at this topic, make sure to read the corresponding article on our website. [1] Dive into an application you almost certainly know about, yeast two-hybrid assays, [2] and check this article out to learn how to increase protein expression in plants. [3]

Resources:
1. Top 10 Uses of Microbes in Biotechnology. Available at: https://bitesizebio.com/48446/microbes-biotechnology/
2. Split Ubiquitin Yeast Two-Hybrid. Available at: https://bitesizebio.com/25286/split-ubiquitin-yeast-two-hybrid/
3. P19 to the Rescue: How to Increase Protein Expression in Agroinfiltration. Available at: https://bitesizebio.com/29964/p19-increase-expression-agroinfiltration/

#102 — Fluorescence microscopy images not only look great but also allow us to get a better understanding of cells, structures, and tissues. And confocal laser scanning microscopy lets us construct 3D images from 2D micrographs.

In this episode, learn the basic principles of confocal laser scanning microscopy, how the microscopes work, and some of its applications in bioscience and beyond.

Check out the corresponding article for a handy confocal laser scanning microscope diagram. [1] Learn about the Airy unit [2] and check out our guide to choosing a fluorescent protein for your experiments. [3]

Resources:
1. Confocal Laser Scanning Microscopy Explained In 3 Easy Steps. Available at: https://bitesizebio.com/19958/confocal-laser-scanning-microscopy/
2. Rubbing Your Microscope’s Eyes: A Guide to Optical Resolution. Available at: https://bitesizebio.com/13415/rubbing-your-microscopes-eyes-a-guide-to-optical-resolution/
3. The Ultimate Guide to Choosing a Fluorescent Protein. Available at: https://bitesizebio.com/54287/choosing-a-fluorescent-protein/

#12 — As a follow-up to our episode about ethanol precipitation of DNA and RNA [1], this episode explains the differences between DNA precipitation in ethanol and isopropanol, helping you to figure out which method is the best choice for your experiment.

Read the full article [2] for handy protocol tips, the differences between using ethanol and isopropanol, and when to use each method.

Resources:
1 - https://bitesizebio.com/2007/12/04/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/
2 - https://bitesizebio.com/2839/dna-precipitation-ethanol-vs-isopropanol/

#11 — Fluorescence is undoubtedly one of the most important and useful tools in a biologist’s toolbox. But do you actually know how fluorescence works?

In this episode, discover what the three steps of fluorescence are, and how fluorescence can be used in flow cytometry.

Read the full article [1] for a breakdown of the key points of fluorescence.

Resources:
1 - https://bitesizebio.com/32973/fluorescent-molecules/

#10 — As you probably know, there are different types of ethanol found in biology labs. It's a versatile solvent used in dozens of experiments and procedures, including disinfection, DNA precipitation, and tissue dehydration.

But which type of ethanol should you use for your application? What's the difference between the types of ethanol in your lab? And how do you handle it appropriately?

In this episode, we answer these questions, providing you with the information you need to keep your research producing top-notch results. Read the full article on Which Type of Ethanol You Should Use [1] for links to additional resources on laboratory applications of ethanol.

Resources:
1 - https://bitesizebio.com/13518/which-type-of-ethanol-should-i-use/

#9 — One of the most commonly used cell lines in molecular biology labs is the Human Embryonic Kidney HEK293 cell line.
Listen to this episode to find out all about the history of HEK293 cells and how to work with them.   

Read the full article [1] for more details about working with HEK293 cells

Resources:
1 - https://bitesizebio.com/45489/guide-to-hek293-cells/

#8 — You probably have or will use SDS-PAGE at some point to separate proteins, but do you really understand how this technique works? Knowing how SDS-PAGE works means you can tweak and troubleshoot your technique as well as impress your supervisor and lab mates.

In this episode, we take you through how SDS-PAGE works, including what SDS does, why you need to use a reducing agent like DTT or beta-mercaptoethanol, and the critical importance of the stacking gel.

Read the full How SDS-PAGE Works article [1] to see helpful visuals for how this technique works and access the table showing the protein sizes that different acrylamide percentages can separate.

Expand your knowledge by buffing up on laemmli buffer [2] and get our Guide to Gradient Gels. [3] If you pour your own SDS-PAGE gels, take a deep dive into the chemistry of how gels work and learn how to pour perfect gels every time with our  Simple SDS-PAGE Gel Recipe with 10-Step Casting Protocol. [4]

Resources:
1 - https://bitesizebio.com/580/how-sds-page-works/
2 - https://bitesizebio.com/44540/laemmli-buffer-what-is-it-for-anyway/
3 - https://bitesizebio.com/47184/gradient-gels/
4 - https://bitesizebio.com/59429/sds-page-gel-recipe/

#7 — In this episode, we cover how jellyfish were able to have a huge impact on biology through the discovery and development of GFP.

Using GFP is now ubiquitous in pretty much all fields in biology, and we'll take you through how three Nobel Laureates developed this valuable research tool. [1]

Many of the applications for GFP are microscopy-based, and we'll discuss how you can utilize GFP for translational and transcriptional fusions, FRAP, FLIP and FRET experiments in the lab. [2,3]

Read the full article for more useful links, hints, and tips on using GFP in your experiments. [4]

Resources:
1. Press release. NobelPrize.org. Nobel Prize Outreach AB 2022. Fri. 1 Jul 2022. Available at: https://www.nobelprize.org/prizes/chemistry/2008/press-release/
2. Fun With FRAP! Fluorescence Recovery After Photobleaching for Confocal Microscopy. Available at: https://bitesizebio.com/19946/fun-with-frap-fluorescence-recovery-after-photobleaching-for-confocal-microscopy/
3. Using Flow Cytometry for Fluorescence Resonance Energy Transfer. Available at: https://bitesizebio.com/21935/using-flow-cytometry-for-fluorescence-resonance-energy-transfer/
4. How a Jellyfish Changed Biology: the Discovery and Development of GFP. Available at: https://bitesizebio.com/13390/gfp/

#6 — Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acid preparations in an aqueous solution.

In this episode, we'll bring you up to speed on how ethanol precipitation works, including the importance of solubility, the roles of salt, ethanol, and temperature, plus a few helpful tips on the side.

Read the full article [1] to learn more about the ins and outs of ethanol precipitation and other DNA clean-up approaches.

Resources:
1. https://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/