Biochemistry and Molecular Biology

Hi all, I am a newcomer, i am dinesh. I would like to know, if any of you are working with colorimetric estimation of the following enzymes, carbonic anhydrase, PEPcase and malate dehydrogenase. If so I will be very happy to know the protocol. I have started collecting few articles and your contribution/help will be appreciated. Thank you.

Hello, hope I am in the right forum. I need information resp. literature recommendations on the mixability of micronutrients in raw powder form (plus helper-substances). To explain: I have to take a whole lot of different vitamins, minerals, trace elements and amino acids. I get them either as raw powder or as in capsule from companies like "Solgar". Because I cant swallow capsules etc, I have to open all of them up, mix them in water and drink it. What I really want to do is a make a whole batch of premixed powders each packed in small plastic bags, so that I dont have to go through the procedure every day, but just open a plastic bag into water. From what I …

If in a subtilisin enzyme assay at pH=8.6 and 25C (both optimal), the absorbance of the solution increased by 0.43 @ 410nm in 1 min, what was the rate of the reaction expressed as Δ[product]/min? The diameter of the assay tube placed in the spectrophotometer was 1cm. I'm confused because the question doesn't give any concentrations, so how can i calculate the rate??

I need to study the relationship between PARP inhibitor and transporter in the blood-brain barrier. I need a method. Could anyone give me a suggestion?

I am taking a general biochemistry class at SJSU and I really need a tutor to help me with homework and studying for exams. I live in San Jose and I need someone located in the south bay area. This is an example of the homework problems that I need help with: The X-ray structure of Hb Rainier (a Y145Cβ mutant of HbA) indicates that the mutant Cys residue forms a disulfide bond with another Cys residue in the same subunit. This alters the position of the β subunit’s C-terminal and prevents its participation in a key salt bridge that normally stabilizes the T-state of the Hb molecule. Explain how the following parameters would be altered in the mutant Hb with …

When virus RNA is produced in the cytosol, Dicer (a RNAse III Enzyme) can cut it to short nucleotides (20-25 nt) that are called siRNA (small interfering RNA). This siRNA can be incorporated into the RNA-induced silencing complex (RISC). RISC can now knockdown mRNA that has the (complementary) sequence of the siRNA. My question: Does RISC knocks down every mRNA? Does it know down the "good" cellular mRNA-transcripts or just the "bad" virus mRNA? I mean, why does RNA interference exists at all?

I was just watching an episode of Fringe which was about a former Nazi who managed to design a toxin which he was able to custom-tailor to literally only target individuals who had certain genetic traits. For example, he could tailor it to only target people who had brown hair, or brown eyes, brown skin, cleft chins, etc. In fact, the toxin could even be tailored to target only a specific person via their specific DNA, or only people of a certain lineage. So I was wondering; would this be possible to do even in principle? I mean I know that it is possible to target specific genes, but could a toxin be designed that could actually do what it did in Fringe? And I'm just…

Hi, I would like to compare the Kcat/Km values for two similar proteins. I have six sets of similar pairs for comparison. Is there any statistical method I can use for comparing the values? I would like to make a judgement on whether the difference in the value is statistically significant (e.g. by p value). Thank you.

I am doing some cloning and using Blue/White screening and I was wondering if anyone has ever done this and ran into a problem of having all white colonies. I am using pGEM-T easy system for cloning. My background control is white also, but I don't understand why this would be.

i wanted to know to what extent simulation of biomolecules structure can be accurate and predict true behaviour of these molecules.

If you grew Hela cells for 100's of years in say a swimming pool full of growing medium. what would it grow into? What would it look like?

Hello, I am cloning a large, 10 kb, DNA fragment and I was wondering if anyone has had any experience with cloning large chunks of DNA. Background: The 10 kb DNA fragment has four genes in an operon. I would like to basically take this operon and express the proteins in E. coli so that they can do their thing (i.e. make the product that they make). Anyway, I heard different things from different people as far as what subcloning vectors to use or not use. For example, pGEM-T some people say use some people say that my insert is too large. TOPO vector I understand will work for something like this. My questions: 1.) Wh…

It is well appreciated that Mg2+ stabilizes duplex DNA 80 to 100 fold to as much as 140-fold (1). How ever Promega (2) and other online calculators of primer Tm do not take into account the effects of magnesium on helix stability. I personally ran a multiplex PCR which its annealing temperature is close to Tm calculated without magnesium effect. I used Wetmore and Sninsky (1995) N-N formula with a little change in [salt] calculation, [salt]= [K+] + [NH4]. Now my question is, do we need to consider magnesium effect for Tm calculation of primers in PCR? since doing so raises Tm about 5-10 oC? [PLEASE SUPPORT YOUR ANSWERS WITH PUBLISHED ARTICLES IF POSSIBLE] Refe…

Hello I am trying to amplify and clone a 10 kb DNA fragment and I am currently using a Pfx platinum kit. I can get fragments <2 kb amplified no problem but I can't get the 10 kb product. Anyone know of a good system to amplify large chunks of DNA? I have a high GC organism I am working with also so I have to use enhancers (FYI I guess).

Hi all, I was looking for recommendations for super-sensitive 96-well microplate DNA assays. We would ideally want something to accurately quantify to serve to normalize our cell proliferation assays. In the past, the lab has used a Diphenylamine Assay, but it does not seem to be working well at the moment. We have started looking into other options, but I was wondering if anyone might have recommendations for ease-of-use/best sensitivity possible for DNA quantification. Thanks for any help!

Hi to everyone, I am righting here for a first time and I would like to ask You a question which may sound a little bit of.. stupid maybe but I can't find information anywhere about it...absolutely anywhere... Ok, through the light phase of photosynthesis some events as water photolysis, proton pumping, reduction and phosphorylation occur.So, when the water molecule is photolysed 2 protons and 2 electrons are released \and oxygen too\.At some point of time those particles would reach NAD+ by reducing it...But my question is: For the reduction of NAD+ 2 electrons and 1 proton are needed...So..what happens with the other proton? I know that the protons are t…

What is the effect of multiple freeze-thaw cycles on genomic DNA?

"The most sensitive amplification methods use an enzyme as a marker molecule attached to the secondary antibody. The enzyrne alkaiine phosphatase, for example, in the presence of appropriate chemicals, produces inorganic phosphate that in turn leads to the local formation of a colored precipitate. This reveals the location of the secondary antibody and hence the location of the antibody-antigen complex." Does this imply that the enzyme alkaline phophatase reacts with the antiboy-antigen complex (the entire complex when assuming indirect immunocytochemistry) (the appropriate chemical) and in turn it leads to a colored precipitate?

I'm not sure if I post it here, but her I go. I know there are about 60-80 billion neurons in the human brain, capable of making a trillion connections. But what I would like to know is how many connections are made during a human life time. How many connections are left unused at the end of my life?

Hi everybody, I am trying to digest a couple of reporter plasmids and having some problems with the size of the expected bands. I enclosed a photo of my lastagarose gel. The first lane is a pGL3 plasmid containing LTR-luc reporter nondigested (size 7446 bp), the second one the plasmid digested with Eco RI (band sizes 2722 and4724 and the tird one the same plasmid digested with PST I (4431, 5389 and 958 expected bands). Then after the ladderthere is another undigested plasmid (PNMT-luc reporter) whose size should be 5533 bp, followed by thedigested one with Not I + Xba I (bands 2625 and 2908). First problem is I cannotfigure out why the gel runs so badly. The run is done …

interior of ER Golgi is topologically equivalent to exterior of the cell This exterior.......is it extracellular fluid? Interior of ER is Lumen?

So codons code for amino acids. But during new DNA replication, there are already proteins (enzymes) made of amino acids that sequentially come together to make new DNA. Where do all those enzymes come from (helicase, polymerases, ligases, topoisomerase) in the first place? In order for DNA to occur those enzymes need to be in place first right? Also where does DNA polymerase get its nucleotides to be inserted into the 3' strand? I am a 3' lagging strand. Thanks.

Is pET-3b used for expression? If so how do you induce it? If you don't induce it how does it work? If it is not an expression plasmid what is it used for? thanks

In lab I was doing a His-Tag of FAAH SDS-PAGE and used commassie blue stain geling afterwards this then turned out to have a lot of bands that go all the way down to the front. I believe I messed up some where. I don't know if it has something to do with the gel, purification, or both. Can anyone help me on this? Ill attach the picture. We ran 2 gels and used them separately for the western blot (no number) and the commassie Blue (no name).

Hey guys, I'm not sure if this is the correct topic to post this under, and I apologize if it isn't. For an assignment in one of my classes, I'm asked to design a procedure that will allow me to change the colour of any type of coin through diffusion. The procedure can involve any variables, as long as there is a colour change, and diffusion causes it to occur. Could anybody suggest a way that this would be possible? Thanks a lot!