Biochemistry and Molecular Biology

Hello! I am currently dealing with monoamine transporters (in this case serotonin transporter SERT) and their expression at the synaptic cell membrane. More specifically, I am wondering if there is more explicit data on the expression of SLC6A4 gene for the SERT. I would like to understand how long it takes for a SERT protein to be polymerized and how long it remains in the membrane before it is degraded. However, I haven't even found the right place (database? publications?..) to search for this problem. I am approaching this topic with a rather clinical background, so any hint from a biochemist would help me a lot. Thank you! C

I am currently performing genotyping in mouse tails using Proteinase K. In order to prolong the shelf life of our Proteinase K we wanted to create a storage buffer. One important part of this buffer is 50% (v/v) glycerol. According to all the literature I could find, the pH of glycerol should be about 7-7.5. However, after discovering our Proteinase K still would not keep, I decided to check the pH of all my ingredients. It turned out, our glycerol (99+%) was actually around 3.5. A different batch of glycerol, with a slightly lower concentration of glycerol (87%, diluted with Milli-Q) was even slightly lower at a pH of 3.2. Even stranger, diluting both batches of glycerol…

Hey everybody, can anybody recommend a good introduction (for a political scientist) into the biochemistry of emotions? I'm currently studying conflict situation in the Middle East and I would like to learn more about the "hard sciences" behind emotions such as hatred, fear, etc. e.g. What happens in the human brain when it experiences fear? How are those emotions triggered? (Sorry in advance for asking in such an ignorand way, but one has to start somewhere...I used the usual search-engines to find introductions, but the articles I found where all rather disappointing.) Thanks a lot in advance.

I am currently trying to express a toxic Fab protein using e.coli. Unfortunately, I have to use this expression vector. I can successfully make the Fab protein at the 2ml scale but have not been able to express at larger 50-100ml scale. Details: pComb vector, SB media, Carbenicillan, with and without glucose, IPTG expression system (non T7 driven) and an e.coli strain that has the laq Iq gene to over express the lac repressor. I am a molecular biologist and am new to protein expression in e.coli. Any advice would be greatly appreciated.

I'm at the end of my studies and I'm doing a research project for a couple of months. My project is based on a publication that showed that a certain F-box protein, which is part of the Cullin-RING ligase 1 or SCF1 complex, is the E3 ligase of a certain protein and promotes its proteasome-dependent degradation. I did CoIPs that showed a strong binding of both proteins. However I couldn't detect an effect of the E3 ligase on the protein, neither ubiquitynation nor degradation. I think that the protein I'm working with is not a substrate of this E3 ligase but since I can detect a strong binding I would like to know what effect this E3 ligase on the protein has. I'm ru…

Hi there, I am thinking about cloning an operon of size 4 kb, consisting of three genes, in pET28a vector, under the control of T7 promoter, is it possible? the genes are spaced by 32 and 89 bp respectively.

I am a first semester medical student. I was reading the harper's biochemistry and when I reached to the carbohydrates section I have been involved with a question about enantiomers, related to D and L coding of biomolecules. The book assumed D-glyceraldehyde and L-glyceraldehyde as enantiomers but their aldehyde and CH2OH groups are identical in both forms. So, would you please help me to understand this, or just introduce me some references (shorts are more preferred) that help me to improve my principal knowledge about this problem? Thank you

I am looking for a simple selection marker other than antibiotic or herbicides/tellurite/arsenite/mercuric salts, because present concept of research is that antibiotics or other selection molecules are stress signalling molecules. So I want some non stress and non signal molecule to perform some transposition experiment in bacteria without any signalling bias. Like, I am thinking a energy source molecule (e.g., carbon source molecule) which e. coli cannot use due to lack of initial metabolic enzyme but may be downstream genes are already present, so in presence of foreign initial metabolic gene in the transposon element it will be able to grow in that particular energy s…

Hi, I am currently working on an enzyme experiment involving casein as the substrate. So basically, several protocols state that after stopping the activity with TCA, the solution should be read at 280nm and a tyrosine standard curve is used for comparison. My problem is that other aromatic amino acids can also be read at 280nm. How do I adjust my computation so that I will only be quantifying tyrosine and not the other amino acids?

Using a patented process I have a tiny amount of dried solubilized keratin, which supposedly can then be used to create fibers. My question is how to preform the later step. In basic terms I broke hair down, took out the keratin, added some "stuff" to it, solubilized and dried it, and now I'm trying to restore it back to fiber form; or at the very least, something more solid than powder. Any help would be much appreciated.

What are the pros and cons of using either site-directed mutagenesis protocols? I'm trying to find some primary sources on the topics too. Thanks!

hallo all, I have a problem with understanding the gatewaysystem. I have a vector that I want to turn into a destination (expression vector). Now I have made blunt ends and the reading frame of my vector ends with a triplet of bps. So I have: vector sequence .... TTA and here I want to introduce the gatewaysystem TGT rest of vector sequence. Now, if I follow the invitrogen information it states I should use the reading frame A (for N tag), but I do not understand how the reading frame stays ok if the resulting attb sites are 25 bps long! The genes I want to introduces into the gateway system (between the attb sites) are ORFs , so they start…

How effectively do deodorants mimic pheromones? Pheromones are integral to the propagation of a species but deodorants....? Please express your ideas. Thanks in advance.

i am trying to clone my gene in pTZ. when ever i do transformation in DH5 alpha strain i get nothing on transformed plates. am doing transformation by T/A cloning method. my ligation is ok. my cells are fine. but still cant get results. can any one guide me?

Hello, I am trying to clone a 1.1Kb gene into 5.7Kb plasmid. It seems straight forward but I got no colony. Below is my protocol. Insert preparation: The insert was generated by PCR. RE sites ( BamH1 and HindIII ) were designed on 5' end of primer. I also generated extra 4 bases for efficient cutting. The PCR was fine. I got a sharp band on gel. I cut the gel and purified it. The product was doubly digested ( BamH1-HF and HindIII-HF from NEB in Cutsmart buffer for 15 min ). Then I gel purify it again. Vector preparation: Vector was also double-digested with two REs. I gel purify the DNA after digestion. Ligation: 80ng of vector…

1 EU is defined as the amount of enzyme that will yield product formation rate of 1 micromole per minute. So it seems that EU has the units of amount (mass or moles), as it's defined as "the amount of enzyme which..." . But in the class they told us that under certain conditions, 1 EU=the reaction rate. So now - EU has the units of rate... for me it's like you would ask me how old am I and I would answer: 55 mph! How is this transition made from the defenition of "amount" to that of "rate"??? what's the math behind it?

this is my first post and this might be a bit strange question, and I seem to be the only one having problem comprehanding that but... Is DNA melting temperature (Tm - temperature when 50% of DNA molecules is denatured) of DNA "sequence1" the same as Tm of sequence1-sequence1-sequence1,that is longer DNA molecules with completely the same GC content). The system I am thinking about has heating rate slow enough not to affect longer molecules denaturation, and heat input is not limiting factor (is provided as much as needed in order to assure constant temperature increase)? The way I see it, you need energy to denature a bond (2 or 3), for this reason GC conte…

Lets say there is a bacteria X with 10 different types of plasmid in it. And i want to isolate each of the plasmid and identify them. How can i isolate each of these 10 plasmid without contamination? Can it be done based on size, Centrifugation based on the kb of the plasmid? I need to know the way to isolate each plasmid separately.

Hello Researchers and Science Lovers, My problem of today involves mitochondria. Specifically mitochondrial metabolism. I have a compound that i believe disrupts mitochondrial metabolism. However im short of ideas at the moment. I could use oxygen electrode for some predictions but what about proteins..How can i study this "disruption" of metabolism studying proteins involved in it as well? What your suggestion for proteins to look? How can i know this compound disrupts mitochondrial metabolism? Thanks everyone. I hope i get some answers.

Before my aunt died of cancer, she performed an experiment with geraniums and water boiled using a jug and a microwave. A control plant was used also which was watered with tanked rain water. 1. Control watered with rain water from a poly tank grew with signs of distress. 2. Same water as above only boiled in a jug, allowed to cool then used to water plant 2 3. Same water as for control, only this time it was boiled in a microwave oven, used to water plant 3. Plant 1 and 2 grew normally, plant 3 showed signs of distress within 2 days and was dead by day 7. Conclusion: unable to draw a conclusion as the experiment has not been repeated, the death of plant 3 could hav…

Hi I wondered if anyone knows how I can make GTP from ATP, or the other way around? Thanks!!

Hi, Apparently, the human body can only absorb about 65mg of vitamin c when taken orally, making mega dose (1000mg) tablets nothing more than a money spinner for drug companies. Vitamin C is an interesting vitamin to me and I am wondering if one were to inject via i.v. 1000mg of vitamin c, would more of it be absorbed and would it be dangerous to inject 1000+mg of vitamin c? [vitamin c is available as a non prescription injection for horses] Regards, Warren

When a person is sick his immune system is compromised and his physical wear and tear is increased. Is this analogous to a more disordered state as evidenced by an increase in entropy? That is why terminal illness precipitates death faster. Please advise.

I am collecting evidence, even anecdotal, how does single nucleotide deletion or insertion in primer region affect the outcome of real-time PCR. I am most interested in how much there is a delay in Cq (treshold cycle value) if compared to perfectly matched primer. But if you do not have Cq data, I also collect "Yes or No" answers, saying, whether there was amplification or no amplification. I am interested specifically in insertion or deletion mismatches. (For substitution mismatches, there is a lot of data.) I will appreciate any published or unpublished evidence.

Hi there, I'm trying to find a spectrometric way to measure Fe(IV) in solution... can anyone suggest a good way to do this?