Biochemistry and Molecular Biology

We are purifying a bacterial dehydrogenase from E. coli cells using a histidine tag. We purified the same dehydrogenase from different bacteria previously, and we have a few examples from the literature. The dehydrogenase from this organism is at least 100-fold lower in specific activity, perhaps more than that. We have a little experience with nickel columns, but not a great deal. Could a small amount of nickel ion be inhibiting our protein? We will try an experiment in which we incubate the enzyme with DTT shortly (there is an essential cysteine). What are other possible explanations?

Hello! I'm new to cell culture, but I need to cultivate the Mle-12 cell line. For three months, I haven't been able to get more than 5-6 passages. With each passage, the number of dead cells increases. This is very frustrating, and unfortunately, I don't have any colleagues who can help me. Could you please tell me what's critical for this cell line and for lung epithelial cells in general? Are they more susceptible to overexposure to trypsin or mechanical stress? My protocol: 1. Pour off the old culture medium. 2. Wash with PBS (3 ml). 3. Add 1.5 ml of trypsin and wait 1 minute. While I wait, I tap the sides of the culture medium, otherwise the cells do not detach. …

AbstractThe Viragenesis Microenvironment (VME) is a distinct, omics-defined biological niche where persistent viral influences cause long-term disruptions in a host’s immune, genetic, structural, and energetic systems. In simpler terms, it’s the place in the body where a virus leaves behind a lasting biological footprint that keeps causing problems, even after the infection seems to be over. This paper defines and characterizes the VME using the Synergistic Compatibility Framework (SCF), a system that identifies fault layers and therapeutic targets across biological domains. Through this lens, the VME is shown to be more than just a byproduct of infection—it is a chronic,…

Are there any regulatory guidance's explaining requirement of type of testing required for Biosimilars marketing firm?

Hi I was reading an article on Science Daily about the discovery of a Lichen in the Mojave Desert, USA. This lichen has developed the ability to resist UVC radiation via a pigment. This got me thinking that I think most things biological can be traced back to genetics, so perhaps there is a genetic change that has taken place so this pigment develops. (probably not quite the right terminology there). in which case could it be possible to map the GENOME and indentify any dna / genes that are responsible for the pigment, then use CRISPR to spice these in to perhaps a plant that we can make polymers from (and make the polymer UVC resistant, or perhaps even some foods, so tha…

I love microscopes, so this really caught my eye. There is little to no information on these guys online, other than a few very old articles. Does anyone have any experience with one of these? How much do they even cost?

In the past I followed a protocol based on Methods in Enzymology Volume 174. We warmed the tubing a bicarbonate/EDTA buffer to about 80 °C. We rinsed several times with warm DI water. We stored in the presence of a preservative, often sodium azide. Is this similar to what other labs do? EDT Does anyone have experience with SnakeSkin dialysis tubing? It seems to be much less expensive than the alternative, although that may be because of a promotion.

We are following a paper in which they purified their histidine-tagged protein (same protein as ours) using a gradient of imidazole. However, they did not specify the volume of buffers. This affects the steepness of the gradient. What would be a logical choice? A Methods in Enzymology article from around 2000 by Barnhorst and Falke Reference suggested a 20-column volume gradient.

We just tried the "Enzyme Kinetics" software module for the first time on a Cary UV/VIS spectrophotometer, which is separate from the Kinetics module. We are plotting absorbance of NADPH versus time. One of its features is that it can calculate the slope of the trace just after one adds the final component to the cuvette holding buffer and all other components. The user defines the time period, which is the linear portion, and the software provides the slope (the units of the slope are a little bit mysterious). This feature could save a good deal of time when one has multiple runs, but there are a couple of software...curiosities. The first is that the software asks for t…

Please, I need help. I bought a synthetic gene from Genscript which came already cloned in a expression vector, pET28a. I'm experienced in protein overexpression, I'm following all standard methods for protein overexpression. Other proteins are been overxpressed normally at my lab, however this particular one isn't. I double checked the sequence, the codons are optimized and everything looks fine. However, the protein does not express at all in BL21(DE3) background. Any clue or suggestion?

What is the difference between lipid nanoparticle than say very small lipid particle? I read that lot in biology articles people making reference to lipid nanoparticle or people making reference to very small lipid particle. What is the difference between the two of them? Also in medicine they make reference to lipid nanoparticle.

I found a paper which expressed the same protein from the same organism as we are trying to do. They uses BL21(DE3) star, and a pET-based plasmid. What struck me is that their conditions for expression (10 µM IPTG; 23 °C for twelve hours) are quite a bit different from any that I have previously used. In their paper they discuss two proteins, and they indicate that they chose conditions to minimize inclusion body formation and to increase yield of protein. I can see the logic in not making inclusion bodies; this protein exists as a dimer, and the monomers are around 35-40 kilodaltons (I have refolded small, monomeric proteins from inclusion bodies, but I don't think t…

I left the field of protein biochemistry to pursue a project in synthetic chemistry some years ago, and now this project is veering into protein expression and purification. We ordered two plasmids from Genscript, and they arrived in the form of what appear to be stab cultures. Therefore, we need to move forward with deliberate speed. I have a basic understanding of sterile technique, but my skills are rusty. What are reliable sources of information regarding handling of E. coli? Now, we need to store our strains, which arrived as stab cultures. In the future, we may need to work out details of the purification of our enzyme, because it is coming from different organism…

Can a viral infection kill you on its "own". Or does it not kill you, but the secondary bacterial infection kills you. I would say the , viral infection can kill you without the bacteria. But would like if anyone has time for a short detailed explain anger. Thank you your time. June,

Formose is a solution made by heating formaldehyde with calcium hydroxide. It's a complex mixture of sugars with various configurations and chain branching, plus minor amounts of alcohols, acids, and other miscellaneous compounds. It's deadly to animals at 20%+ of the diet, with 25%+ killing all of them, and causes them to lose weight and have moderate to severe diarrhea. Other anomalies observed include shrunken livers and spleens and swollen kidneys and adrenal glands. At 10% and lower it actually causes higher weight gain than pure glucose solution, yet they still have mild diarrhea. Purified formose solution enables longer survival than crude formose. Now, let's specu…

How many colours can see a Tetrachromat in a Rainbow ???... ( millions of colors !!!.... is NOT an assesable answer !!!... ) Humans with three(3) /cones\ !!!... can see in the rainbow seven(7) colors !!!... Humans with two(2) /cones\ !!!... can see in the rainbow three(3) colors !!!... ( BLUE-WHITE-YELLOW) !!!... Humans with "only" one(1) /cone\ !!!... can see in the rainbow "only" WHITE !!!...

Imagine the soul as a curious traveler who needs a vehicle (your body) to explore and experience the universe. Your body's cells are like a sophisticated vehicle ; functional, complex, and capable of interacting with the environment. The soul doesn't "operate" these cells, but uses them as a means to perceive and interact with the universe. Without functional cells, the soul is like a traveler without a vehicle present but unable to move, see, or experience anything. When cells become dysfunctional or die, the soul loses its ability to interact with the physical world. Think of it like this: Cells are the car Soul is the passenger Universe is the land…

Hi, I was looking for plant biology podcast, and I discovered than there was really few Podcast on plant science (specially with a molecular point of view). 1) Do you have any idea why plant science seems to be so forgotten ? 2) Do you think it can be interesting to create a podcast about it (on photosynthesis, plant immunty...) 3) Why should be important for something like that ? I hope this topic is in the right place. Thanks

Hello I thought that eating meat and lentils did provide the same proteins and same effects, then I heard about PDCAAS Could you please suggest some mix/alliance of vegetable proteins to obtain the same result/quality of animals proteins? Regards

I have cast and used SDS PAGE with Tris/glycine/SDS electrode buffer. However, I was planning to use some precast, gradient gels for the first time. I am trying to contact the manufacturer, but I have not yet heard back. "BisTris" is in the product description, and the catalog page mentions the possibility of ordering MES or MOPS buffer. My first question is whether I should change my recipe for the sample loading buffer. My old recipe includes Tris (pH 6.8, same as stacking gel), SDS, 2-mercaptoethanol, glycerol, and bromphenol blue. My second question is what sort of power settings should I use? In the past I have typically used about 100 volts during t…

How to Extract Pollen DNA from honey using Promega Wizard Purification Kit?

Are all E-Coli Strains Really Harmless? Debunking the Common Myth Many people think that when they eat raw meat, drink raw milk or even a bad piece of lettuce, they will be infected with Escherichia coli, more commonly known as E. Coli, a bacteria strain. According to the CDC, when people get E. Coli, they are prone to diarrhea, urinary infections, pneumonia, sepsis, and many other illnesses. Because of the terrible effects the bacteria strains bring, many people rightfully believe that all strains of E. Coli are dangerous…but are they? Most E. Coli strains are harmless and benefit a person’s intestinal tract. These strains help people digest food, produce vitam…

We are running a 15% PAGE here, and at the end these line start to appears. First we thought that it has something to do with too much pressure or something like that, but one time we had an eletric oscilation and the line had a curve... Anyone know how to solve it? the image represent the end of the gel, down below the last band imagem_2024-09-13_184128695.pdf

Hello, I would like to express a protein using a cell free protein expression kit from New England Biolabs. The protein is a chymotrypsin and so it must be activated through cleavage to be functional. I am trying to understand the best way to express this protein. I am not sure if I express only the sequence of the activated protein if that would cause problems (e.g. from my understanding the zymogen form can sometimes help fold properly). Alternatively, I could express the inactive form and hope it becomes activated in my assay (I will be applying it to some tissue in which it is normally found). Or, since chymotrypsins are typically activated by cleavage by trypsin…

Good evening everyone, I'm working with sequencing of tRNA, applying the mim-tRNA Seq protocol (Behrens et al., 2021) but i don't understand the step of dephosphorylation, in which T4 PNK is employed... I know for sure that this step is crucial for adapter ligation on the 3' end of RNA, since when we have tried the ligation without a preliminar dephosphorylation step of total RNA and de-acylation step of tRNA, the ligation was failed. This enzyme has a 3'-phosphatase activity, but tRNA presents a 3'-OH end so, why is this dephosphorylation step important? I supposed that maybe during the de-acylation step (using Tis-HCl) a phosphoryl group remains attached on the 3'-…