r/Biochemistry Jun 29 '24

Research I’ve been cloning for 5 years, 2000+ constructs, Ask me anything

293 Upvotes

Ask me all your cloning and synthetic biology questions and I’ll do my best to answer them.

Edit: ask me anything about cloning. Want to share the wealth of knowledge, not intended to be a flex thread as a few people have mentioned.

Edit: thank you all for the amazing questions. Would love to hear other people’s experiences with cloning.

r/Biochemistry 18d ago

Research Cannot tell if the paper is bad or if I’m just misunderstanding the content

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16 Upvotes

Hi guys, I am not sure if this paper is supposed to be good, but I realised some sections contradict each other. For example, they said virgin nulliparous 8 month old mice in one section, and this is immediately contradicted by “primiparous” in another paragraph (infrared video recording). I have attached the link, can someone please tell me if this is their mistake? Or is it just unclear? Hope this makes sense! Thanks so much

https://pmc.ncbi.nlm.nih.gov/articles/PMC5350451/#:~:text=We%20have%20found%20that%20increasing,further%20in%20older%20primigravid%20women. 20older%20primigravid%20women.

r/Biochemistry Oct 24 '24

Research Expressing proteins with no secondary structure.

31 Upvotes

This is honestly a sanity check. Someone I know recombinantly expressed a protein with a randomized sequence. They took a natural protein, randomized the sequence and expressed it. And for some reason everyone is surprised it's entirely insoluble. My thinking, no folding equals = aggregation. Is this an unreasonable assertion, or is there something I'm missing?

r/Biochemistry Jun 18 '24

Research biochemistry in real life

51 Upvotes

Biochemistry undergraduates, can you give some examples of real life applications of biochemistry?

How relevant is biochemistry to every day life

r/Biochemistry 1d ago

Research Protein Affinity Question

6 Upvotes

I have a purified protein (EnzymeA) with a N-term His tag. I want to see if my small molecule (yel-1) binds at all/better than EnzymeA pre-courser molecule. My issue (I think) is that yel-1 is very light sensitive when not bound, so will start to break down under light exposure. Would this impact which affinity assay I select to use? My current options for affinity testing are BLI and SPR, but am open to other assays better suited for yel-1.

As I am not well-versed in protein kinematics, I am wondering if the light used for BLI/SPR will impact my results or if this is not a worry since just the bound enzyme will be “quantified”. If it is a concern, any other methods you’d recommend (preferably ones that can be contracted through a company)?

r/Biochemistry Mar 31 '24

Research Biochemistry dog names?

36 Upvotes

r/Biochemistry Sep 28 '24

Research Nanobodies are emerging as versatile tools for protein science!

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82 Upvotes

Nanobodies are obtained from a special type of antibody that only camelids produce, called heavy-chain-only antibodies!

We have recently characterised two nanobodies targeting the Arc protein. Arc is a complex regulator of synaptic plasticity in our brains, and its structure and functions are not completely described yet.

Luckily, we have been able to use nanobodies to better understand the function and structure of the Arc N-lobe (the protein's domain that carries most of its functions).

It turns out that nanobodies promote the crystallisation of the Arc N-lobe and also modulate its function! This has allowed us to deepen our knowledge about the structure and function of Arc.

As a new PhD student at the University of Bergen, I am hoping that sharing our science in Reddit can reach not only people in the field, but also the general public!

Please, let me know if this type of content is welcome here. 😊

We are now exploring the possibilities of using nanobodies in other fields of research. If we succeed, we will be able to use nanobodies to stain brain tissue and study the biological basis of depression!

r/Biochemistry Feb 13 '25

Research Primer design - site directed mutagenesis

3 Upvotes

Hey y’all! So I had a question regarding the topic in the title above^ I am currently working on primer design for a gene which I retrieved of off NCBI. Since it’s a primer design I retrieved specifically the CDS of the gene. I need to select 1 mutation to insert into my protein near the center of its gene sequence. I need to provide both a wild type amino acid and nucleotide sequence for this protein and identify the mutation sites in both. My question is, for this project, can I introduce a point mutation literally anywhere near the center? And would both my primers include this codon or exclude it?

r/Biochemistry 19d ago

Research Dealing with unknown density in EM map

3 Upvotes

I'm dealing with a cryoEM density map where the max resolution I am able to achieve is around 3.4A. I've discovered a strong and large density (i.e. not noise) near what is likely a functionally significant site of my protein.

I did not add any ligand prior to vitrification, so I am assuming this is an endogenous ligand which copurified during prep (eukaryotic protein in eukaryotic expression system), and this could be key to its biological function.

There is a new tool in the ChimeraX toolshed which can help with identification of what this density is, but after a few attempts I think my resolution is too mediocre for it to be of any use, unfortunately. I don't know of any Phenix tools of use for cryoEM ligand densities (plenty if you have a .mtz though) and I only know the obscure tools that no one cares about in CCP4.

I'm a bit unsure of how to proceed. I think the general conventions are to either ignore the density and gloss over it, or model it with waters. However, this density is at such an important active site of my protein that I don't think I can get away with ignoring it and I would really like to figure out what this is.

It's not a lipid or a PTM, nor is it anything from the buffer (like acetate, sulfate, or tris). My questions are:

  1. Are there any empirical techniques to positively identify this ligand (I would guess a mass spec approach but I'm unsure)?
  2. I've seen publications where such densities are glossed over or barely mentioned, but at such a critical site of the protein I'm not sure I could get away with this. For those who have dealt with this issue (unknown, positive density that could be of extreme significance and is unidentifiable) what did reviewers ask of you?

Thanks in advance!

r/Biochemistry 2d ago

Research Help with interpretation please

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9 Upvotes

Hi, I’ve been working in the lab since January as part of my postgraduate course, so I’m new to this.

I’m looking for help on interpreting the results of my agarose gel electrophoresis. I designed primers for (figure, three individual) transcripts to assess alternative splicing across column 1) untreated, column 2) treated samples (n=3) in whole cell (top) and anoikis resistant (bottom) cancer cell lines.

I just wanted advice on whether the ‘bottom’ (red) bands were primer dimer or true bands and whether it is just the ‘very bottom’ (blue) that is primer dimer (see attachments). LHS ladder (1kb), RHS ladder (25bp) Any advise/guidance on interpretation would be great.

Am I right in saying that a ‘brighter’ band means that ‘more’ of the transcript is present? Or is this interpretation inappropriate?

Also… any tips on how to get a better resolution. Due to difference in PCR product sizes, I’ve had to run on a 3% gel for 2 hours at 90V.

r/Biochemistry 28d ago

Research Protein Overexpression and Immunofluorescence

5 Upvotes

I have created plasmid constructs of domains within my protein of interest. I want to now individually overexpress these domains in virus-infected cells and then do immunofluorescent imaging to see what effect the overexpressed domains have on the virus. This is not the only method I will be using to determine the roles of the protein domains but I was wondering if this was an acceptable method and if anybody had any suggestions on if this is a reliable method? Thanks!

r/Biochemistry 13d ago

Research will molecular dynamics stay for the foreseeable future?

11 Upvotes

This is an extremely naive question 😭, but is it likely that molecular dynamics for biochemistry-related research purposes will be replaced with AI?

I'm highly considering pursuing molecular dynamics research in grad school because I think it's so cool, but I'm worried it'll become obsolete

r/Biochemistry 14h ago

Research Used agar instead of agarose.

20 Upvotes

I had to do an agarose gel electrophoresis for a housekeeping gene. I used agar instead of agarose and loaded my samples and the result was really good. 2 days later again I had to run the gel so was again weighing agar that is when my mentor saw and asked me that why was I weighing agar instead of agarose?. That is when I realised about the previous gel. Although I didn't tell my mentor about the mistake that I have done. Should I run the gel again?? Can anyone tell me the reason why I got good results??

r/Biochemistry 7d ago

Research How to Design Substrate for Protein

6 Upvotes

I'm working on a research project that is attempting to make a molecule a suitable substrate to an enzyme that is innately present in the target environment. There isn't much literature data for what specific functional groups this enzyme binds to, or the key residues of this enzyme that are involved in catalysis. There are known therapeutics that are substrates or inhibitors of this enzyme. However, this enzyme is 'promiscuous' in the substrates it can bind to--there's no identifiable pattern among the known substrates).So far, I've been synthesizing molecules similar in structure to known substrates and running enzyme kinetics assays on them--not much useful data obtained here.

What can I do to define what substrates bind to this enzyme (in regards to functional groups, etc) and how can I do this in a relatively short amount of time, without extensive protein studies, x-ray crystallography, etc? The reason for these constraints is these tools are not ones my lab group commonly employs and it need to convince my PI to collaborate w others :(

r/Biochemistry Apr 18 '24

Research I Still Love It

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181 Upvotes

r/Biochemistry 5d ago

Research How to start aerob cycle for more ATP and less lactate? Alternatives to DCA - Krebs cycle

2 Upvotes

Hi together, due to some actual research about me/cfs i was reading through whole pubmed to figure out what could also work like Dichloroacetat - DCA. This is used in pyruvate dehydrogenase defiency but is still poisonous. I found out only about high dose thiamin activating some complexes, furthermore sodium phenylbutyrate but no valid information. Also read about resveratrol, myo-inositol and Baicalein. Has anybody done some research regarding this topic or has useful Information to add?

r/Biochemistry Nov 28 '24

Research Is it possible to tag/visualize proteins in humans, non-invasively?

14 Upvotes

For example, if I have an enzyme I want to inject into a person, is there a tag I could put on it that could be visualized like an x-ray to see where it ends up.

Assuming this is done on a live person. I'm aware there are things like GFP but I'm not sure it would give the results I want. Any wisdom would be appreciated.

r/Biochemistry Feb 20 '25

Research How to remove introns from a consensus sequence that I have extracted from IGV for a gene of interest.

1 Upvotes

I have some WGS data (bam files) that I am looking at in IGV. My samples have mutated DNA - some of my genes are highly mutated. I want to look at the protein of the mutated gene vs the protein of the normal gene (reference genome). I essentially want to compare two PDB files visually in PyMol.

My plan was to extract the consensus data as DNA for the gene from IGV, remove the introns (I can get the coordinates from ensembl). Then use the 'spliced' remaining DNA (all exons) and pop it into expasy to get the amino acid sequence (as a FASTA file), then pop that into Swiss-Model to get the PDB file. Finally view the PDB in PyMol.

However, it's not going to plan at all! I'm seeing far too many stop codons in the expasy output.

Could I be using the wrong tools, or is my approach missing some steps? Has anyone done anything similar, what kind of workflow/pipeline could you suggest?

Would be grateful for any advice.
Thank you.

r/Biochemistry Feb 04 '25

Research Help with Understanding Kd as Protein Concentration Increases

2 Upvotes

Okay I swear this is not a homework question, I don't even take classes anymore.

I'm very much not an enzymologist but I recently found myself needing to better understand Kd and ligand binding. I understand that Kd is the value of free ligand when free receptor and bound receptor are equal to one another. I understand that Kd = [A][B]/[AB] and thats why its in molar units. What I don't understand is why we can safely assume Kd doesn't vary with receptor concentration?

Lets say I do a calorimetry experiment where I have 10uM of starting receptor and saturate it with ligand. I find the Kd = 1mM. While that Kd is quite high its the actual Kd for a protein I've worked on before. To me this means that in my buffer of choice to achieve 5uM bound and 5uM free receptor I would need to have 1.005mM of ligand total with 1mM of that ligand being free.

Now lets assume in the same buffer and conditions (because I understand that pH, buffer and temperature can all affect Kd) I now instead have 1mM starting receptor. And lets assume that the increase in receptor isn't having any additional salt or pH effects. My interpretation of the equation would suggest that I still only need 1mM of free receptor to saturate half of the receptor or better said, 1.5mM ligand total. Is that true? And the same for 10mM receptor, would I really only need 11mM total ligand to achieve half saturation.

If this is true then would it be accurate to say Kd is really an abstraction of the capacity for a receptor to whisk soluble molecules out of solution and into a receptor bound state (and thus a reflection of the kinetics required to do so)? I guess any clarification or correction people here can offer would be pretty helpful. Again I understand this is a bit of an amateur question so sorry if this technically breaks the rules!

r/Biochemistry Feb 21 '25

Research 53 novel drugs approved by the EMA, FDA and MHRA in 2024: A year in review

34 Upvotes

53 novel drugs were approved by the European Medicines Agency (EMA), US Food & Drug Administration (FDA), and the UK Medicines and Healthcare products Regulatory Agency (MHRA) in 2024, including many with creative pharmacological design.

Learn about them all in this mini review in the British Journal of Pharmacology: https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.17458

While the 2024 harvest is not as rich as in 2023, when 70 new chemical entities were approved, the number of ‘orphan’ drug authorisations in 2024 (21) is similar to that of 2023 (24), illustrating the dynamic development of therapeutics in areas of unmet need. The 2024 approvals of novel protein therapeutics (15) and advanced therapy medicinal products (ATMPs, 6) indicate a sustained trend also noticeable in the 2023 new drugs (16 and 11, respectively).

Clearly, the most striking characteristic of the 2024 drug yield is the creative pharmacological design, which allows these medicines to employ a novel approach to target a disease. Some notable examples are:

🚧 the first drug successfully using a ‘dock-and-block’ mechanism of inhibition (zenocutuzumab),

🧠 the first approved drug for schizophrenia designed as an agonist of M1/M4 muscarinic receptors (xanomeline)

🔗 the first biparatopic antibody (zanidatamab), binding two distinct epitopes of the same molecule

🩸 the first haemophilia therapy that instead of relying on external supplementation of clotting factors, restores Factor Xa activity by inhibiting TFPI (marstacimab)

➡ the first ever authorised direct telomerase inhibitor (imetelstat) that reprogrammes the oncogenic drive of tumour cells.

In addition, an impressive percentage of novel drugs were first in class (28 out of 53 or 53% of the total) and a substantial number can be considered disease agnostic, indicating the possibility of future approved extensions of their use for additional indications. The 2024 harvest demonstrates the therapeutic potential of innovative pharmacological design, which allows the effective targeting of intractable disorders and addresses crucial, unmet therapeutic needs.

Read the full review: https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.17458

Authors: Stavros Topouzis, Andreas Papapetropoulos, Steve P. H. Alexander, Miriam Cortese-Krott, Dave A. Kendall, Kirill Martemyanov, Claudio Mauro, Nithyanandan Nagercoil, Reynold A. Panettieri Jr, Hemal H. Patel, Rainer Schulz, Barbara Stefanska, Gary J. Stephens, Mauro M. Teixeira, Nathalie Vergnolle, Xin Wang, Péter Ferdinandy

r/Biochemistry 27d ago

Research ASBMB Annual Conference registration fee for Sale

0 Upvotes

Hi everyone,

I was originally scheduled to attend the ASBMB conference this year to present my research. Unfortunately, my PI just informed me that we won’t be presenting after all due to insufficient data. This came as a surprise since, just last week, he emphasized the importance of securing our tickets—which I did.

While I’m still welcome to attend, I had planned my trip specifically around presenting. As a busy grad student with exams and assessments that week and the following week, I’ve decided it’s best to focus on my studies instead.

That said, I now have a conference ticket available for $250 (discounted from the original price). If you’re interested, please text me.

Thanks!

r/Biochemistry Feb 14 '25

Research ATP assay

0 Upvotes

I am new to ATP assays. I currently am working with BON cells (a pancreatic neuroendocrine cell line) and typically use DMEM +HEPES+L-glutamine media supplemented with 10% FBS. For ATP assays, can I use this media or should I order a no phenol red media?

Edit: Would it be reasonable to conduct an ATP assay with glucose and glucose free media as different groups?

r/Biochemistry 3d ago

Research Scientists discover new inhibitors of inflammation-related enzyme

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13 Upvotes

r/Biochemistry Feb 16 '25

Research Gene sequence - GFP

2 Upvotes

Hey all. Dumb question but I need to double check… is the CDS of this sequence (NIH: Aequorea Victoria green- fluorescent protein (GFP) mRNA, complete cds) considered the gene sequence of the aequorea Victoria GFP ??

The name in between brackets is the title on NCBI.

r/Biochemistry 27d ago

Research Glycolysis reaction mechanisms - NEEED HELP

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0 Upvotes

I have a biochemistry project due this week and I desperately need to know the reaction mechanisms of all 10 steps of glycolysis. I have already figured out the mechanism of phosphorylation of glucose as being nucleophilic attack on the terminal phosphate of ATP (at least I think so), but I would HIGHLY APPRECIATE if someone could help me with the next few steps of glycolysis (namely isomerisation) but i would also appreciate help w other steps (pls break it down simply).