International Microorganism Day 2022 – How to Get Involved

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Uncategorized

Though they may seem small and insignificant to the everyday person, as researchers we know that microorganisms are a critical part of our everyday life. As we observed with the recent coronavirus pandemic, studying these microbes is important for creating cures and treatments. Microbes are used in a variety of ways that benefit the environment and in valuable processes such as food production, so understanding microbes is an important field of research.

Here at Azure Biosystems we value and support microorganism research and provide equipment and reagents that can aid in research involving microbes. Let’s celebrate International Microorganism Day, happening this year on Saurday, September 17, 2022!

Bak, a mascot for International Microorganism Day
Bak, a mascot for International Microorganism Day

What is International Microorganism Day?

International Microorganism Day (IMD) was started to highlight and celebrate the essential role that microbes play in our everyday lives. In honor of Anton van Leeuwenhoek’s discovery of the first microbe. Since its beginning in Portugal in 2017, the organization has greatly expanded and now partners with universities and event venues to hold events all over the world.

The mission behind IMD, according to their website, is to “to teach and provide a platform for microbe lovers around their world to share their passion and knowledge.”

4 Ways to Get Involved with IMD 2022

With numerous ways to participate, microbe lovers everywhere can get in on the conversation and share their knowledge and love of microbes. We’ve put together 4 easy ways to join in on the fun:

1. Attend an in-person event near you

Events are happening around the world, like Japan, Germany, Portugal, and India, to name a few.

  • Brussels, BelgiumMuseum of Natural Sciences, Saturday, 17th September 2022, 10am-6pm
  • University of Catania (Italy) – Saturday, 17th September 2022, 9:15 am – 13:30pm Register here
  • University of Dhaka (Bangladesh) – Saturday, 17th September 2022, 10am-5pm – Registration link here
  • Sweden – Uppsala Antibiotic Center – Saturday, 17th September 2022, 10am-1pm – Registration link 

To see if there is an event being held near you, check out their website for the most up-to-date list.

2. Tune in for one of the online events

Don’t see an event near you? Don’t fret! Many different organizations are holding different events for IMD, including live streams. Podcasts, like Microbes and Us by FEMS, will be doing a special episode. Simply do an internet search for IMD online events to see the many options.

3. Create your own event

This is a great opportunity for science outreach in local schools or even the local community. There are many interested in microbes that aren’t working in a scientific capacity, and this would be a great way to connect with them and bring more awareness to the importance of microbes. For ideas on how to put together an event, the IMD organization has put together an event handbook that can be found here.

4. Join the conversation on social media

Look for the hashtag #InternationalMicroorganismDay and follow @IntMicroDay anywhere on social media to see the events going on and the conversations already happening around microbes for the event. Happy International Microorganism Day!

To find more information about how Azure Biosystems can help in your research of microorganisms, check out our website.

To learn more about the International Microorganism Day 2022, check out the IMD’s website by clicking here.

6 Tips for Troubleshooting Poor Band Separation on SDS-PAGE Gels

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SDS-PAGE Transfers Troubleshooting

Poor separation of protein targets is one of the most common and frustrating issues with associated with Western blotting.  To troubleshoot the problem, we must first understand how protein separation by SDS-PAGE works. SDS-PAGE stands for sodium dodecyl sulfate (SDS) Polyacrylamide gel electrophoresis (PAGE). It is the universal first step in denaturing Western blots. The goal of SDS-PAGE is to separate proteins by their molecular weight.

How do you separate proteins using SDS-PAGE?

Polyacrylamide gels form a crosslinked, mesh-like matrix through which proteins migrate with the application of current. The matrix functions like a sieve, allowing smaller, low molecular weight proteins to migrate quickly through the gel, while large, high molecular weight proteins tend to progress more slowly. When the electrophoresis is finished, proteins in each lane will be separated into a continuum, with the highest molecular weight proteins remaining near the loading site and the lowest molecular weight proteins reaching the opposite end of the gel.

An important caveat to this rule is this: proteins only separate exclusively by size if they have been denatured prior to being run on a gel. All proteins have inherent tertiary structures related to their biological functions. Such structures confound the process of separation by molecular weight alone. Proteins of any size with globular structure tend to migrate slower through the gel matrix, giving them a similar migration pattern to proteins of higher molecular weights. Such proteins can easily be misidentified if identification is based only on molecular weight.

Because of this phenomenon, it’s important you remember to denature your protein samples prior to separation by SDS-PAGE to remove higher levels of structure. This step also allows the proteins to be separated almost exclusively by molecular weight as they migrate through the gel matrix. SDS detergent, the denaturing agent DTT, and boiling are all used to disrupt higher structure and linearize proteins.

Many proteins also have an inherent negative or positive charge, which can disrupt uniform migration driven by electrical current, as in gel electrophoresis. SDS used for denaturing also serves to coat the proteins with an overall negative charge, allowing the proteins to migrate in response to electric current without influence from their natural charge. As denaturing reduces the confounding effects of protein structure on migration, so uniformly charging reduces the effects of native charge.

6 Suggestions to How to Improve Separation of Bands by SDS-PAGE

With an understanding of how SDS-PAGE facilitates separation of proteins by molecular weight, it is possible to identify areas of the process that may contribute to band separation issues. Here are 6 ways you can start troubleshooting:

1. Ensure proper sample preparation

If the proteins in a sample are not denatured properly, they will not migrate through the gel as expected. You have to ensure the amount of SDS and DTT used to denature the sample is appropriate. Try slightly increasing the boiling time to make sure all samples are completely denatured (although boiling too long can degrade proteins, so finding an appropriate boiling time is key). About 5 minutes at 98°C is common for samples prepared with denaturing loading buffer.

After boiling, place the samples immediately on ice to prevent gradual cooling prior to loading. Allowing the samples to naturally return to room temperature will undo the denaturing step, as most proteins will renature if allowed to cool slowly.

Loaded gel in electrophoresis for SDS-PAGE
Protein samples loaded into a gel inside Azure Aqua Vertical Gel Running System

2. Load the appropriate amount of protein sample

Loading an excess of protein per well can cause the proteins to aggregate during electrophoresis, preventing them from separating by size and causing them to run together. This can result in clustered bands that cannot be individually defined. Additionally, excess protein can bleed into neighboring lanes, preventing sharp lane distinctions.

For best results, use the minimum amount of protein or cell lysate required for downstream detection. It is always necessary to validate each protein-antibody pair and it may take multiple tests to determine the optimal amount to load for each protein of interest. On the other hand, loading too little protein will result in faint or undetectable bands.

3. Make sure your polyacrylamide gel is fully polymerized

The polymerization of the polyacrylamide gel is important for efficient electrophoresis. Make sure the gel has had enough time to completely polymerize before using in PAGE. Incomplete polymerization is often caused by forgetting to include key ingredients, especially TEMED. Double check you have added all the ingredients, that they’re fresh, and added in the correct concentrations.

If your budget allows, purchasing pre-made polyacrylamide gels can be a simple solution to these polymerization issues.

4. Change the polyacrylamide gel percentage

The percentage of polyacrylamide used in a gel affects the number and size of pores in the matrix. Gels made with high amounts of polyacrylamide have small, tight gel matrixes; those made with low amounts of polyacrylamide have larger, more spacious matrixes. Proteins of all sizes migrate more quickly through lower percentage polyacrylamide gels and more slowly through higher percentage gels. Because of this, gel percentage should be chosen based on the size of the protein of interest.

High molecular weight proteins require gels with a low percentage of polyacrylamide. If the matrix is too small, these large proteins will be not be able to migrate efficiently and will stay grouped together near the loading site. Similarly, low molecular weight proteins require high percentage gels for effective separation. In a low percentage gel, small proteins tend to migrate too quickly. The open gel matrix allows small proteins of a range of molecular weights to migrate together, resulting in poor band resolution.

Because these pores are the reason certain proteins migrate through the gel, using a gel made with the appropriate percentage of polyacrylamide for the protein of interest is very important. The higher the percentage of polyacrylamide, the harder it is for proteins to move through the gel. This can be helpful if you are wanting to separate proteins that are small. If you are using a higher percentage gel on larger proteins, they cannot fully separate because they won’t be able to move through the gel.

If proteins of interest are relatively small and are not spreading, try using a higher percentage of polyacrylamide gel. If the proteins of interest are larger and are not spreading, try a lower percentage gel.

ADDITIONAL RESOURCE: Bendy Bands and What to do with Them

5. Use fresh buffers

The buffers used for running SDS-PAGE require very specific salt concentrations and other components to allow the current flow properly and the proteins remain denatured. Protein separation can be hindered by overused or improperly formulated buffers. It is good practice to make fresh buffers before each run or as frequently as possible if large volumes are used regularly

6. Adjust the electrophoresis parameters

Finally, gel temperature is affected by the amount of current applied to the gel and can affect the migration patterns of proteins. Generally, the gel should be prevented from getting too hot. A simple way to address this is to run the gel at a lower voltage for longer time. With the Azure Aqua Transfer Cell, you’re able to perform quick, 1 hour transfers or transfers overnight at lower voltages. Alternatively, the gel can be kept cool by placing a compatible ice pack in the buffer chamber or by placing the entire gel apparatus in a cold room while it runs.

Conclusion

We want to see your Western blots succeed! Band separation is key for successful SDS-PAGE and downstream applications, such as Western blotting. Issues with band separation can occur, but the six troubleshooting tips provided here will help you solve the issues and return to collecting data quickly.

Here at Azure Biosystems, we are dedicated to bringing you quality products that will help you run flawless experiments and obtain quality data every time. Check out this page to make sure you have everything you need to run a successful Western blot. Be sure to use the form on this page to ask for help!

NEXT READ: Trouble-free Transfers

 

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3 Tips for Preparing Western Blots for Publication

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Imaging Western Blotting

Western blotting is a tried and true way to detect and evaluate protein expression and is widely used by researchers. While it has been around for decades, Western blots are still presented as data in both scientific talks and in published manuscripts. Getting a Western Blot that is ready for publication is the goal but as many scientists can attest, it is not always easy. The 3 tips in this blog post will help you prepare your images for publication.

There are a number of steps in the Western blot process and many of these contribute to getting Western Blot images that are publication-worthy. For instance, if you do not have adequate blocking, then you can end up with lots of background noise. While you may essentially see the answer you have been looking for, if the image of the Western blot is not ideal, it can bring into question the validity of the result all together.

3 Tips for Publication-worthy Blots

1. Thoroughly Plan Your Experiment

We went into this in more detail in this article, but planning is key to having optimal Western blot images. Steps like validating the antibodies being used and ensuring all materials are not out of date can help with the overall success of the experiment. A big part of planning that affects publication is the order of your samples. Make sure you are loading the samples in a way that would be ideal for publication. For example, if you have extra lanes, you may consider adding in some additional samples to simply test if they too have a protein you will be probing for, but these samples have nothing to do with the experiment at hand. This can be a wise use of resources, but if done incorrectly, can jeopardize the chance of your Western Blot being used in a publication.

If you choose to add extra samples, add them at the end of the gel preferable with an empty lane separating them from the other samples. This allows you to easily remove them from the image for publication. If they are in the middle, then you will be forced to digitally cut them from the image, and it can raise suspicion when you have clearly pieced together the Western blot image. To avoid this altogether, be mindful of the order of samples.

 

2. Get a Variety of Exposures

A key part of getting publication-worthy Western blot images is the exposure and how the image is acquired. As a graduate student, I heard countless times about how I did not get the correct exposure of the Western blot. It took me a good while to figure out that it is really important to get a variety of exposures from faint to over-exposed. Having a number of exposures not only allows you to fully assess the data, but it also gives you a number of options for use in a publication.

If you’re using film, this is important because the way it looks on film may not be conveyed once you scan it so you want to have options to choose the one that best represents your data. This is one of the biggest pros of using an imager, like the Azure Sapphire. If you’re using an imager, then the imager will inherently capture a number of exposures and images for you to use from and this ultimately saves you a lot of time.

Instead of having to stand in the dark room doing a variety of exposures by hand at various lengths of time and hoping you chose the right ones, you can capture it with an imager in a matter of minutes and have a number of exposure options to choose from for your publication.

3. Label and Organize

This may seem obvious, but not all scientists are naturally organized and orderly, so it warrants being said. Being organized and having all things labeled is really important. Even if you do not think the experiment is usable for a publication or a talk, you never know if it will be needed later. With this in mind, always make sure to take the time to label each lane (right on the film if you did not use an imager), add the date, and make it very clear which experiment the exposures go with. You will likely perform many Western blot experiments. It can take years to get the data needed for a paper. Instead of trying to figure out which Western blot images go with which experiment you did 2 years ago, save yourself the time and headache by making sure everything is organized and labeled well the first time.

Another tip when creating figures is to make sure you denote which Western blot image was used in the figure, including the data and exposure chosen. There are many times when you may put together a figure for lab meeting or a poster at a conference, but even if it is not for a publication, make sure to note which experiment it came from. You may end up using this figure in the manuscript after all. This will save you time and stress in the end to have these details already determined.

With these 3 tips, you will set yourself up to get publication-worthy Western blot images every time. Incorporating these tips into your Western Blot routine will prepare you for success when it comes to time to publish your data. What are your favorite tips? Share them with us using the form on this page!

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