But you don’t *always* want a lot of branching because it doesn’t make for very sturdy structures. Imagine you have a pile of glue-covered sticks you want to bundle together to hold up your tent. If the sticks are branched it’s hard to get them close together and you have less points of contact for the glue to go to work. But if your sticks aren’t branched, you can easily stick them together. The biochemical equivalent of this is at play in structural carbohydrates. Instead of glue, you have lots of -OH groups which can form weak partial-charge-based interactions.    The difference can be dramatic - compare cellulose and glycogen. Both are made up of just glucose, but cellulose is unbranched and β-1,4 linked. This linkage allows to to form long straight chains that can stick to gather for sturdiness.     For even better stickiness, many structural carbohydrates have modifications like amidation and oxidation which provide additional binding opportunities. For example, chitin, a structural carb found in fungal cell walls & the exoskeletons of insects & crustaceans (crabs, etc.) has the same linkage as cellulose, but instead of plain glucoses it has N-acetylglucosamines. This makes chitin super strong & stiff.     But “structure” doesn’t have to mean “stiff.” Some sugars play structural roles by forming gels. Those -OH groups love water, and water loves them, so sugars can “soak up” water to form gels. Depending on how much sugar vs. water and what type of modifications the sugar has, these gels can have different properties. In the lab, we use the sugar agarose to make gels we use to separate DNA pieces by size through “electrophoresis” (using charge to get the molecules to travel through the gel). That’s a really easy way to see sugar gels at work, but they’re also at work in our bodies, forming things like the cushioning around the bones in our joints & the bouncy-ball-ness of our eyeballs!    A lot of the time, sugars work together with other molecules in “glycoconjugates” where sugar chains (glycans) get attached to non-sugar things. And this teamwork is accompanied by team names that can be confusing…    The order of the names of these “hybrids” indicates which is dominant - the first one is the minor component and the last one is the main component. So, glycoproteins are proteins with sugar chains attached; proteoglycans are sugars with a sprinkling of protein; peptidoglycans are sugars with some peptides helping link them together; and glycolipids are sugars linked to the lipid molecules making up membranes. They have a lot of diversity and, correspondingly, a lot of diverse functions. Here are a few examples.    Glycoproteins     This is where you have the protein-ness dominating, with sugars attached. I know I said “minor” but some glycoproteins can actually be “mainly” sugar - ~1/2 of all proteins have at least one glycan & some glycoproteins are up to 60% carb by mass. Glycoproteins are often found in the outer leaflet of the plasma membrane, facing the cellular exterior, or secreted into the extracellular matrix.     The sugar chains can be O-linked or N-linked. O-glycosylation is where a sugar chain gets added through the -OH group of a Serine (Ser) or Threonine (Thr) protein letter (or a hydroxylated lysine or proline). N-glycosylation is where a sugar chain gets added through the amine group of an Asparagine (Asp) letter.     In addition to needing to use different enzymes, O- & N-glycosylation differ in several important ways. In O-glycosylation, individual sugars are added one at a time, but for N-glycosylation, the same “starter tree” gets added in the beginning of the protein processing process at spots on the protein containing the consensus recognition sequence Asn-X-Ser/Thr, where X is any amino acid other than proline (proline’s that awkward-shaped amino acid whose side chain swings back around and attaches to the backbone making things tricky). Throughout the protein processing steps, this tree gets whittled down to a common core, which can then be added onto.     This whittling might sound kinda pointless and energy-wasting - I mean, why add something just to remove it, right? But it serves as an important quality control measure and makes sure that the protein gets directed to were it needs to go in the right order, etc. Unlike proteins, sugars are *not* genetically encoded - only the enzymes responsible for making, placing, and processing the sugars are. So our cells rely on the selective expression of these, their compartmentalization to different membrane-bound protein-processing centers inside of cells (e.g. Golgi bodies), etc. to make sure that the “right” sugars get added in the right places.

For example, proteins bound for secretion have a signal sequence that directs them to a membrane-bound room within the cell called the Endoplasmic Reticulum (ER) while it’s being made. From there it gets passed through a variety of compartmentalized pouches of the “Golgi body” and those compartments have different modifying enzymes that can trim & modify it. Since all of this is happening inside of membrane-bound rooms, the protein never has to really see the inside of the cell, so it can be optimized for the environment it’s gonna face when it gets released.     If a protein isn’t folded properly, it won’t get whittled correctly so it won’t get permission to go to the next compartment and, if it can’t get its act together, it will be directed to the protein shredder (proteasome) instead. But if the whittling goes well, it can get modified and then those modifications can direct it different places to get modified in more ways and/or get released outside the cell or onto the cell surface.     Depending on which “versions” of the modifying enzymes a person has, they can make slightly different sugars. This is the case with the ABO blood groups, which refer to the sugar groups displayed on the surface of blood cells. There’s this glycosyltransferase enzyme (sugar adder/mover) made by the ABO gene. People with type “A” blood have (2 copies of) one version of it that adds on an N-Acetylglucosamine. People with type “B” blood have (2 copies of) a different version of it that adds on a galactose. People with type “AB” blood have 1 copy of each of those so they make and display both. And people with type “O” blood have a total dud version of the enzyme that can’t add on any sugar, so they only display the core pentasaccharide (5 sugar) part (H antigen).    The reason this matters (and how it was discovered) is because these sugars can serve as antigens (things that antibodies recognize). People who have type O blood won’t be used to seeing A & B antibodies, so if you give them blood with A and/or B antigens, they’ll see that blood as foreign and attack it. Similarly if you give type A people B and/or AB or if you give type B people A and/or AB. This is why we say AB is “universal recipient” and O is “universal donor.” note: Rh factor is the (+/-) thing and it involves a different protein. also note that the ABO sugars can be attached to lipids to anchor them to the cell surface as we’ll discuss, or they can be attached to proteins and secreted, but not all people have the secreted version which is why CSI shows sometimes talk about secreters vs. non-secreters.  more on blood types here: bit.ly/abobloodtypes   Proteoglycans    Here, we flip around the starring roles - with proteoglycans, the sugars get the starring role and the protein just serves to kinda hold them all together. These sugars are GlycosAminoGlycans - large polysaccharides with a lot of modifications. These modifications include lots of amino sugars (hence the A in GAG) but they also often include things like oxidation to carboxylate or the addition of sulfate groups. GAGs can have different patterns of modifications that serve as a sort of code that is “read” by proteins in the extracellular matrix (ECM) (the molecularly-rich environment in between cells). How it works is that the different modifications make for different binding sites that complement different proteins’ structures. So then those proteins can bind, which can be useful in several ways. Sometimes, binding may get the proteins to shape-shift (undergo a conformational change) that activates or inactivates it. Other times, binding might serve to just keep the protein there. And, since GAGs can be really long, they can bind multiple proteins and serve as “hubs” or “scaffolds” at which that multi-protein reactions can take place.    In addition to those more specialized functions, proteoglycans have “generic” ones. Modifications like carboxylation & sulfation add negative charge. And when there’s negative charge, you know metal cations (positively-charged molecules) are gonna wanna come play! Sodium ions (Na⁺) drop in to serve as counter ions, and they bring water with them. So these proteoglycans soak up a bunch of water, gel-like. Different ones (which include heparan sulfate, chondroitin sulfate & keratin sulfate) have different gel-ness-es & thus are best suited for different things - from tense tendons & strong nails, to the shock absorbers between your joints, & the mucus in your nose! Yup, “mucus” is a proteoglycan consisting of a mucin protein covered with sugars! There are actually dozens of mucin proteins and the proteins involved in mucin & in all the other proteoglycans vary a lot - which they can do since it’s the sugars starring here!

Peptidoglycans    Here, sugars get an even bigger role. Peptides play supporting roles (literally) - short chains of amino acids serve as bridges between carbohydrate chains for extra sturdiness. These are seen in bacterial cell walls, and many antibiotics target them. More on that here: bit.ly/penamp     Glycolipids    We’ve talked about how a lot of the sugars coating our cells are anchored onto glycoproteins embedded in the plasma membrane (the phospholipid lipid bilayer surrounding each cell). Another way our sugar trees can get anchored to the cell surface is by attaching directly to the lipids themselves, and these are what we call glycolipids. There are several different subclasses of them with different names (of course…) These include lipopolysaccharides, glycosylphosphatidylinositol (GPI), cerebrosides, & gangliosides.     Lipopolysaccharide (LPS) is a glycolipid present in bacteria’s outer membrane, with different bacterial species having different sugar combos in their LPS. These sugars provide Gram-negative bacteria (ones w/o a strong cell wall) with a barrier that makes it hard for would-be attackers to get to the membrane itself (and potentially into the bacteria). So it serves to help protect the bacterial membrane from things like antimicrobial molecules and viruses. But not humans - our immune system recognizes LPS and attacks it. May sound great, but too much LPS can cause septic shock (a sort of immune system overreaction when bacteria are in your blood). You might have heard of LPS by another name - endotoxin - just like that nickname suggests, LPS is toxic to our bodies and is responsible for some of those symptoms of bacterial infections people experience - this includes fever (LPS is a fever-inducer aka pyrogen). To prevent over-reaction we have enzymes that can break it down    Glycosylphosphatidylinositol (GPI) is a short sugar chain that serves to anchor non-membrane-spanning proteins to the outside of the cell. One end of the sugar links to a phospholipid head and the other end hooks up (through a phosphoethanolamine linker) to a protein. So, with a GPI-anchored protein you have lipids, carbs, & proteins all working together! Molecular teamwork is so cool! Our bodies use a bunch of these proteins to do a bunch of different things including helping cells recognize each other and acting in signal pathways.     Cerebrosides & gangliosides are glycolipids that I’m not sure what do but there’s a bunch of them in nervous tissues & if there are problems making or breaking them you get issues.     Wrapping up our sweet science…    One thing I learned (or relearned since I probably did learn it in undergrad but then forgot) is that glycogen (that’s the animal storage carb remember) is attached to a central protein called glycogenin, which actually is a glucosyltransferase that does the pioneering work of adding on the first glucoses (before passing the job over to glycogen synthase & a branching enzyme).     Having glycogenin as a central hub allows glycogen to form granules that can chill in the cytoplasm (general cellular interior) until called upon. The main glycogen storage locations are the liver and the muscles. When blood sugar levels drop, glycogenolysis starts in the liver and the glucoses newly freed travel through the bloodstream to tissues in need! (your brain & red blood cells are a couple of the main reliers on this process).

I had always thought of glycogen as just tangles of sugar, and apparently most scientists had too. Because when a scientist named Bill Whelan discovered glycogenin people didn’t believe him but he used his biochemistry brilliance to prove ‘em wrong!    Whelan served for a time as president of the International Union of Biochemistry and Molecular Biology (IUBMB) which is a great note to end on because was originally one of my weekly “broadcasts from the bench” for them Be sure to follow the IUBMB if you’re interested in biochemistry (now on Instagram @the_iubmb)! They’re a really great international organization for biochemistry.    This week I’d also like to thank Professor John Tansey, a chemistry, the Director of Biochemistry and Molecular Biology at Otterbein University in Ohio. I first met him at an ASBMB conference where he caught my attention because he was there with undergrads and he was so excited for and supportive of them. And this warmed my heart so much because a lot of profs at big institutions sometimes overlook undergrads and undergrad education is hugely under-attention-paid-to at many places. This is one reason I really want to become an undergrad professor at a small school and why I love being able to help teach undergrads (and everyone else following along) through this social media/blog stuff. Anyways, Dr. Tansey kept in touch on Twitter & when he wrote a biochemistry textbook he sent me an advanced copy (no strings attached). And it’s really good - and its where I learned a lot of what I told you today (glycobiology wasn’t covered that much in my undergrad classes). The book is published by Wiley & called “Biochemistry: an Integrative Approach”     here’s more about Carolyn Bertozzi’s pioneering work on bioorthogonal chemistry: blog: bit.ly/bioorthogonal_prize ; RU-vid: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-LrwzFRY7gsk.html          more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com                                                 #scicomm #biochemistry #molecularbiology #biology #sciencelife #science #realtimechem

Thank you! I'm grateful this platform allows me to help more people

Happy to (hopefully) help! Best of luck with the presentation and the degree!

Glad it helped!

Glad I could help!

Proteins and RNA absorb UV light, so a UV detector can scan the gradient and absorption peaks tell you where “stuff” is - and there are characteristic places in the gradient you can expect to find monosomes, polysomes, etc. You can detect “global” differences if the ratios are skewed (for example, if “all” translation is inhibited, you’d expect to see an increase in monosomes and a decrease in polysomes).     But the UV can’t tell you what specific RNAs are are in those peaks, so you can take the “fractions” of the gradient (e.g. the monsoonal fraction & polynomial fractions) & look to see what’s where. First you have to get the gradient out - after centrifugation and separation of your monosomes, polysomes, etc., you use some way to push the gradient out from a hole in the top of the tube (old school style by injecting a higher concentration of sucrose through the side of the tube to build pressure from the bottom or with a fractionation with a piston that pushes down from the top to squeeze it up through a hole in the piston. And you can UV it on the way out as you direct it into fractions (kinda like with protein chromatography except you’re taking the column with you and doing it from the bottom).    To do that you have to extract the RNA out of the sugar (often by phenol-chloroform extraction). It’s often harder to extract the RNA out of the goopier stuff (higher density sucrose), so you’re likely to lose more. To control for this, you often add known quantities of a control mRNA, like luciferase mRNA, to each fraction before you start trying to extract the RNA. This way, you can measure how much luciferase was lost during the extraction in each fraction to normalize the fractions (adjust them so they can be directly compared to one another). This way, you don’t get fooled into thinking there’s more of an mRNA in the monosomal fraction just cuz you recovered that fraction’s RNA better.    But how do you know what’s in what fraction? If you have one mRNA in particular you’re interested in, you can see where that recipe ended up in a couple ways. One is by doing a northern blot on the various fractions. A northern blot is where you use electrophoresis to run RNA through a gel mesh which separates the RNA pieces by size. And then you transfer those RNAs out of the gel and onto a membrane and use labeled probes complementary to RNA you’re looking for to see where that RNA is on the membrane. Alternatively, you can use RT-qPCR, which makes a bunch of copies of a region bookended by primers that you give it. So you can use primers specific to a gene of interest and see how many copies get made.     A northern blot or qPCR work well if you know what recipe you’re looking for, but they’re low-throughput and you have to know what to look for. With the rise of high-throughput RNA sequencing methods, it’s now become possible to sequence the RNA associated with the different fractions (e.g. monosomal fraction, polysomal fraction).     Most of the time, when people talk about mRNA-seq, they’re typically talking about sequencing “all” of the mRNAs without addressing whether the RNAs are actually being actively translated. The idea is that if you break open a cell and count the number of mRNA copies of a gene there are, if you see a lot of an mRNA, a lot of its protein is likely getting made - but that’s an assumption that’s not always true. It’s easier since you don’t have to go through all of this ribosome fractionation, and there’s less risk of losing some of the RNA in the process, but you don’t know if that mRNA is actually being used.     This is different from ribosome footprinting. Ribosome footprinting lets you see where along the river the boats are at a certain point in time. Instead of leaving the mRNAs intact, you use RNases (RNA chewers) to cut up the RNA around the bound ribosomes - the region the ribosome is standing on (~30 letters) is protecting from cutting, so then you can release this protected RNA and sequence it to see where the ribosomes were. Since ribosome footprinting chews around the ribosomes, it separates ribosomes that are on the same mRNA (but different locations on it) at the same time. So what you end up seeing is the average of where the boats are in all those copy “rivers.” So you can’t tell if you have 3 boats on the same river or 2 on 1, and 1 on another, 1 each on 3 etc.

But, in the case of polysome profiling, since you don’t mess with the mRNA neighboring ribosomes are on, you do separate “3 on 1” from “2 and 1” and “1 and 1 and 1.” (But once you pass 8 or so on one you can’t tell if there are more cuz they all come out in the same fraction). But you can’t see where on the strand they are - so you lose information about whether certain regions are translated more slowly than others (rare codons causing a holdup?) or whether alternative start sites are being used.    If you want to validate stall sequences, start sequences, etc. and/or determine what’s required for ribosomes to stall there, you can turn to ribosome toeprinting (aka primer extension inhibition).     blog form: bit.ly/toeprinting ; RU-vid: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-CJhQNzHOhLY.html     Ribosome toe printing (aka primer extension inhibition) works by giving ribosomes in a cell-free translation system a template and using reverse transcription with a labeled primer that targets the 3’ end of that template. Let the reverse transcriptase do its thing and it will make a complementary DNA (cDNA) copy of the end of the template until it runs into the ribosome. Then purify those labeled cDNAs and run them alongside sequencing lanes that show you where A, C, T, & G are in the template (you can prepare these by reverse-transcribing the template and spiking in labeled dideoxynucleotide (dead end nucleotides) - 1 letter per reaction - this will cause there to be a range of cDNAs ending in the labeled letter, and you can compare them to your sample. You can do things like add ribosome inhibitors like cycloheximide (CHX) (at concentrations where they prevent elongation but not initiation) to get the ribosomes to build up at start sites so you can see them. And even without adding anything you can see if the ribosome is stalled places already.     Sometimes ribosomes stall because they’re translating awkward sequences like things with a bunch of prolines. And “programmed” stalling can actually be used by cells as a regulatory mechanism. Sometimes stalling is caused or relieved in the presence of various drugs or metabolites (small molecules that are part of metabolic pathways - so breakdown or build-up products). This stalling often occurs in upstream open reading frames (uORFs) and regulates expression of the main ORF (which has the protein making instructions). A cool example of this is some bacteria making an antibiotic resistance gene, ermC in response to the presence of the corresponding antibiotic, erythromycin. More on that here:    Vazquez-Laslop N, Thum C, Mankin AS. Molecular mechanism of drug-dependent ribosome stalling. Mol Cell. 2008 Apr 25;30(2):190-202. doi.org/10.1016/j.molcel.2008.02.026     And here are some other examples of toeprinting at work…    Here’s the original paper: Hartz...Gold (1988) Extension inhibition analysis of translation initiation complexes, Methods in enzymology www.ncbi.nlm.nih.gov/pubmed/2468068    Here’s a paper that uses it in a PURE expression system (where the translation’s happening in a reconstituted system where the minimum required components have been purified and combined) as opposed to a lysate-based system (where you break cells open (lyse them) and use the machinery that was in there, unpurified - and you add some extra factors to make things more efficient). This paper has nice a methods section.    Cédric Orelle, Teresa Szal, Dorota Klepacki, Karen J. Shaw, Nora Vázquez-Laslop, Alexander S. Mankin, Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition, Nucleic Acids Research, Volume 41, Issue 14, 1 August 2013, Page e144, doi.org/10.1093/nar/gkt526     more on PURE systems here: blog: bit.ly/cellfreeexpression ; RU-vid: ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE--yVxvsF8gMM.html     This paper uses it to study stalling at termination codons: Karousis, E.D., Gurzeler, LA., Annibaldis, G. et al. Human NMD ensues independently of stable ribosome stalling. Nat Commun 11, 4134 (2020). doi.org/10.1038/s41467-020-17974-z    Here’s a paper describing a fluorescence-based system, where they do sequencing with a capillary gel electrophoresis sequencing machine instead of reading out radiolabeled bands on a slab gel.     Egorova, T., Sokolova, E., Shuvalova, E., Matrosova, V., Shuvalov, A., & Alkalaeva, E. (2019). Fluorescent toeprinting to study the dynamics of ribosomal complexes. Methods, 162-163, 54-59. doi.org/10.1016/j.ymeth.2019.06.010     Here’s a paper about a different technique, inverse toeprinting, which goes at things from the 5’ end with sequencing so you can vary the template randomly to see what sequences might cause a stall, and then see what caused that stall.     High-throughput inverse toeprinting. Britta Seip, Guénaël Sacheau, Denis Dupuy, C Axel Innis. Life Science Alliance Oct 2018, 1 (5) e201800148; DOI: 10.26508/lsa.201800148 www.life-science-alliance.org/content/1/5/e201800148   more on ribosomes: bit.ly/rad_ribosomes more on my postdoc work with mitochondrial ribosome profiling: bit.ly/postdocprerprint Context-specific inhibition of mitochondrial ribosomes by phenicol and oxazolidinone antibiotics Brianna Bibel, Tushar Raskar, Mary Couvillion, Muhoon Lee, Jordan I Kleinman, Nono Takeuchi-Tomita, L. Stirling Churchman, James S Fraser, Danica Galonic Fujimori bioRxiv 2024.08.21.609012; doi: doi.org/10.1101/2024.08.21.609012 more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

You might want to try altering concentrations of protein, volume, etc. And/or sending out for high-throughput screening if you can afford it. Good luck!

Great to hear!

Sorry but I'm not clear on your question

@@thebumblingbiochemist sorry! I was very confused myself but I think I might have found a way to untangle my problem 😊 thank you as always!

Glad you got it figured out!

I think this will help with your first question bit.ly/wherestheprotein ru-vid.com/video/%D0%B2%D0%B8%D0%B4%D0%B5%D0%BE-jTQxHK3o3lE.html not sure about the # of His - guessing just convenience

@@thebumblingbiochemist Thank you very much!

FastCloning is very similar, just more temperamental (but easier)

@@thebumblingbiochemist I appreciate the info!

So glad to hear. Best of luck!

So glad I could help!

Good luck!

Correct. We added the CAS several days later.

@@thebumblingbiochemist thank you 🙏

Thanks! The volume depends on the size of the well and gel thickness so I can't really be much help with that. Gel loading tips, slow, right into the bottom of the well is best. I don't know re formamide vs. urea. I typically used formamide, but urea in the gel. I typically just used markers of known size (purchased individually, then mixed). Best of luck!

@@thebumblingbiochemist Gotcha okay thought i was going to have to do that for the ladder -- theyre just the typical biorad 15 well combs with 1mm thickness if that helps in answering that question Also ive seen that TTE (taurine instead of boric acid could help), have you ever tried in your experience?

I would probably try to stay 5 μL or below ideally. And no I haven't tried that. Good luck!

ChimeraX is definitely a great alternative and I am in no way trying to dissuade anyone from using it. But we're using PyMol in my class and I'm therefore making content to help my students that I'm therefore sharing more widely as well. But thanks for your perspective

Glad it was helpful! Thank you! Best of luck with school!

Thanks so much!

Sounds like you might have some ethanol or something still from the kit. Be sure to spin the column well, including a final dry spin. Good luck!

We've been taking a sample from the starter culture as our "pre-induction" sample. And, you really just have to try it and compare! Good luck!

Sorry, but that's something that - as you suspect - you've got to try and see! Different proteins like different things. Good luck!

Thanks so much! Best of luck with your experiments!

You need to use cells that have T7 with the lac promoter like BL21(DE3) and then put your protein under T7 - so it makes T7 that then transcribes your protein.

more on t7 based expression here: bit.ly/bacterialproteinoverexpression

Hi - I'd be worried that the different conditions might influence the staining

@@thebumblingbiochemist oh ok great point! Thank you so much 😊

Great to hear. Thanks!

Thanks so much! Glad to help

Glad I could help!

You're very welcome!

thank you!

Thank you! So glad it was useful!

Thank you so much! That means a lot to me to hear

So happy I could help and so sorry you feel lonely

Thank you so much! Hope this can help: ru-vid.com/group/PLUWsCDtjESrHU5vdztBYlQ4hK3iiccSnH & bit.ly/adobe_illustrator_fundamentals

Thanks! Your first explanation

@@thebumblingbiochemist thank you!

Soooo happy I could help! Best of luck on your exciting journey!

Happy to help!

Thank you! Happy I could help - best of luck to you as well!

@@thebumblingbiochemist Thank you so much Brianna! ❤🙏

Primary - cuz it leads to all the rest!

Thank you so much! Happy I could help!

I think we got it from Sigma

@@thebumblingbiochemist What is sigma? Is it a site? Can I ask if international shipping is available?

It's a brand/supplier

That's so great to hear. Best of luck!