Biochemistry and Molecular Biology

plz tell me if i want to put a reaction with NHS ester of arginine and glycine..what solvent i hv to used and why??

Probably Molecular Biology 101, but I was wondering if anyone could tell me how an amino acid's pKa can be altered by protein environments and also by surrounding charge? e.g. Glutatate and aspartate can hold onto protons around the physiological pH in some instances (microenvironment and surrounding charge) when their pKa is much lower - how do they do this!

I've remained at a fairly constant weight for a very long time even though I'm eating _lots_ of fatty foods (McDonald's, macaroni with cheese... or more like cheese with macaroni, sausage, pizza...) and don't really exercise at all. My calorie intake is sometimes a bit above what it should be and with the amount of fats I'm consuming I probably should be quite overweight by now. My metabolism is rather fast but not extreme and definitely shouldn't overcome my diet and lack of exercise by itself. However, I normally eat a very large meal around 5h before I go to sleep. I tend to sleep quite long (more than 12h sometimes) and don't really like to eat until I've been awa…

I'm interested in small scale PCR, but I'm just a layman in this area. I got some questions regarding the components needed. I start with the dNTP. dNTP-mixes (or the individual dATP, dCTP, dGTP, dTTP) are often sold in quantities of around 0.5ml with a concentration of around 10-100mM. As I got it, quite often you only would like to use 50ul with a concentration of around (say) 50-100uM per vial. If I do large scale PCRs I would probably dilute and use up most of the dNTP-mix I bought. But if you're doing small scale PCRs with say just 1-2 vials at the time, how is the dNTP handled/stored, since it is not wise to freeze/melt dNTP more than a couple of times? Is …

please give me some idea that why only RNA primer used in DNA replication ?

is it possible that we can isolate DNA on water instead of gel?

how will you know mol wt of a DNA sample without running marker...just with help of tracking dye.......

hi! All I was using an old mac and all was well , then I moved to this new intel mac and I cannot run seqman and editseq which is rather important program for my work. I mostly do site directed mutagenesis and used these programs for sequence alignment to check for mutation, IF YOU GUYS KNOW ANY ALTERNATIVE TO THIS PLEASE LET ME KNOW THANKS RN

Hi All, I'm trying to find concentrations for NADH and NAD+ in the cytosol of humans and plants, better if it's Arabidopsis thaliana. I've been led to believe that in humans the total NAD(H) concentration is between 3-500 uM, depending on the state of the cell, of which 80-85% is NAD+. I'm coming up empty as far as numbers for plants go. Anyone know or have some advice? Justin

Apparently the Indy gene, a Krebs cycle intermediate transporter, is expressed abundantly in fat body, midgut, and oenocytes. To me, this suggests that there's a locus controlling region regulating expression, but I haven't seen this reported anywhere. Would it be safe to assume that this is true, or are there other gene regulation mechanisms which could also be at play?

Has anyone else here heard of Epigenetics? Essentially, what has to do with instead of altering the code itself, it alters the chromatin, which in turn silences or less a gene go full blast. When the chromatin curls up the gene becomes silenced, when it is loose, the gene becomes heavily active. The great thing about Epigenetics is, although it is less permanent, it is heridetary, and controlled by your environment, and especially various types of nutrients. I believe(I think I am not the only one who believes this, a few, VERY few obscure scientist may think this also) that chromatin may be in a transitional state when notch signaling, a cellular signalin…

Hi, I'm having problems in b-galactosidase stability during luciferase assays. Typically we transfect cells (different cell lines) with Lipo2000 in 24wp using 0.15ug pGL3 luciferase-reporter gene; 0.1 ug SV40-bgal (or 0,025 ug pCMV-bgal) plus 0,3ug of our different repressors of interest (some of them also under pCMV control). We made equal the final amount of DNA/well with pBSKTT gene. We transfect them during 4-6-12h and change the medium and analyze by luminometryafter 48h . We observed good luciferase signals (low background, 200-300.000 RLU). We measure b-gal adding corresponding substrates (sometimes the half of the indicated reactive) and after 1h of incub…

I need to pick some brains: We have been using OVCAR-3 cells in our lab for years. All of a sudden, they are dying after a few days in culture. This happens a few days after trypsininzation and reseeding. The culture conditions are the same, the media are all the same (fresh). The other cell lines are doing fine. We have received new aliquots from ATCC and it happens. This also happens to cells that we thaw from cryopreservation. It's very frustrating because this has been going on for some time and has caused havoc. Any ideas?!

if u see human body as a machine,its income energy and use the energry,its seems it can work forever.

This is the scenario: At least a 2 dozen boxes of crumbled medication (it was a very brittle presentation (soluble in the mouth) was contained in a room- there was lots of dust of this medication everywhere. It was cleaned and thrown away but......can the dust left behind, in non-visible to the eye from SO MUCH medication, stay somehow penetrated in the environment or objects in came in contact with? And still produce some for of effect on highly sensitive people?

Cameron Alexander on his recent talk (and subseqent paper in Nature) on sythetic biology. He also talks a little about presentation style at the big conferences. http://www.test-tube.org.uk/videos/pages_cameron_bald_guy.htm

Is it possible for a medication to leave "nano"-particles (smallest sized particle invisible to the eye) penetrated in objects? And if contact with them (the particles), somone who is hiper-sensitive in general and to that particular medication, still feel some residue effects??? Please someone highly knowledgable on this topic solve this mystery!

I'm trying to determine Kd values for protein:RNA binding using tryptophan fluorescence. Buffer: 10mM HEPES (pH 7.5) 100mM NaCL 4mM MgCl2 1mM TCEP [protein] = 0.25uM; RNA is titrated in 0 - 4uM. Instruments used: -Perkin Elmer LS 55 -Varian Cary Eclipse Fluoresence quenching data is corrected for the inner filter effect ad fit to a quadratic equation. Problem: Im measuring Kd's in the nM range for my protein:RNA complex, but when I titrate RNA into BSA or lysozyme as negative controls, I also measure nM Kds. Same goes for my protein:poly-dIdC. This is very confusing. Why would I be measuring such tight binding amongst molecules that should h…

Hi, I am wondering whether anyone has any information on the enzyme pyrophosphatase and the conditions which affect its activity ie pH, temperature, enzyme concentration..I am finding it hard to locate this information in journal articles. Any help would be great Thanks

Silly question, I know. But I just have to know...and they just don't teach us this stuff at school...

Here's a new way of making PNA antisense oligonucleotides: Make artificial tRNA synthetases --http://www.cnrs.fr/cw/en/pres/compress/EvolutionCodeGenetique.htm -- that attach to PNA nucleotides, such that the one that hooks up to "A" in the mRNA sequence will place "U" in the PNA sequence. Of course, you'd have to add the 3' and 5' UTRs after the fact --in the 3'...5' format, of course--, so you could prefab those PNA oligonucleotides, and splice them to the oligonucleotide. Automatic antisense oligonucleotide manufacturing!!!

Hi There, I am optimising an enzyme assay that measures the appearance of NADPH. I began by creating a standard curve ([NADPH] (mM) vs. ABS@340nm) for NADPH in the buffer that I intend to use to identify my lower and upper detection limits. I performed it in triplicate and got nice tight data (all STDEV were less than 10% of each point - in most cases less than 1%). The linear portion of the graph has an R2 value of 0.9981 (5 data points). The only problem is that the slope is only 4.98, but it should by around 6.022, as this would be the correct millimolar extinction coefficient for NADPH. Can variables like temperature or buffer composition change the extinction coe…

I believe I may have posted this before on a slightly less appropiate subforum, so I'm moving it here. Your expert opinion is greatly needed!!! Synthesize a peptide nucleic acid antisense oligonucleotide for HIV’s gag gene’s mRNA. The reason for using PNA is that it is substantially more resistant to enzyme degradation by nucleases and proteases. The reason for choosing the gag gene is that it’s proteins, p24, p17, p7, and p6, code for the basic physical infrastructure of HIV; w/o these key proteins, there is no HIV. Then, encapsulate the oligonucleotides in liposomes studded w/ anti-CD4mAbs. This will ensure 1) toxicity is limited—cf Ambisome, the liposomal prep…

The amino acid histidine has a side chain for which the pKa is 6.0. How would I show what fraction of the histidine side chain will carry a positive charge at pH 5.4?

when i do the plasmid extraction by using published method by Oloni Kotchoni et al. (2003), i didn'y get any band when analyzed by 1% agarose gel...but when i measure by spectrophotometer, the concentration of the 'plasmid' is quite high, ~1000ng/ul and the purity is quite good... but then, i do PCR colony juz to check the insert of my cDNA..but, i still didn't get any band in the gel picture... actually, this is the 2nd time i do the electhroporation..the 1st time i do it by using same method, i was successfully obtain the cDNA...but the 2nd time, i fail to observe the cDNA in my plasmid... so, any suggestion @ advice for me.... p/s - is there any effect…