Biochemistry and Molecular Biology

Okay, I'm extracting RNA from whole blood samples. I've done a red cell lysis and got my RNA extracted via a modified version of the Chomsinsky method. The problem is that I'm co-extracting far too much DNA with the RNA. I ran the samples down a gel, and I've got the 28S and 16S rRNA bands as well as a lot of genomic DNA contamination. I've run a second extraction against phenol:chloroform. I've just added the isopropanol and there's an aggregate forming instantly, which to me screams of DNA contamination. I've not DNase treated the samples yet because there seemed to much contamination to get rid of it all sufficiently. I need to get this extraction really …

How would one go about, practically, probing a pool of glycoforms (isoforms) of a particular protein for the type which are most biologically active, that is to say the ones which have the greatest activity? The "brute force method," so-to-speak, would involve assaying every single one and if the potential population is very large then you can imagine how costly and cumbersome the task would be. Much insight could be gleamed from a bioinformatics approach, but I'm wondering does anyone know of a particular approach in the literature? Or does anyone have any suggestions?

Hi, I am trying to measure CoA levels in HepG2 and Hek293 cells using a recycling enzymatic assay (Allred and Guy 1968). Has anyone done this as I am having difficulties obtaining reproducible results? Also I think I may have enough CoA in my samples to measure accurately. Many Thanks, Pascale

Guys I've searched and searched but I havent found what I've been looking for. What are the individual roles of the different amino acids found in enzymes, i.e. how do they aid catalysis. I know the basic Nitrogen of histidine is used to abstract a proton from serine, threonine or cysteine to activate it as a nucleophile but I dont even understand how that helps in catalysis! Not to talk of the other 19 amino acids. I need help people. Thanks.

And what about in cell membranes? Is it the same case there? (Just need to quickly finish my practical report...lol).

Dear All: I really need your help with this, please reply as soon as you read the massage !!!!!!!!!!!!!!! I read about the L-H PCR (Length Heterogenecity PCR) which is an amplification of 16S rDNA gene and separates it based on the length of the gene. The question is how the 16S rDNA gene is having different length and how this works with the primers. I amplified 16S rDNA gene using 27f and 1492R primers and when I ran them on a gel they all lined up in one raw having the same length. I am totally confused!!!! please help me out with this if you know any thing about it.

can anyone explain the principles of lipid extraction (chloroform:methanol , Foltch method) ?? thanks.

Hi, I'm trying to get the article 'Electron microscopy of prefibrillar structures and amyloid fibrils' (1999) by Nielsen et al. in the journal 'AMYLOID, PRIONS, AND OTHER PROTEIN AGGREGATES'. My university has recently changed its journal access system, I can't get it. Would anyone be able to attach this article (or similar ones, i.e. reviews of the use/technique of electron microscopy in amyloid fibril analysis) to a reply? Thanks.

Hello all... I'm taking a course covering, amongst other things, simple enzyme kinetics. I'm fine with the majority, but am not quite sure of the physical significance of the catalytic proficiency and catalytic efficiency, given by [math]proficiency = \frac{k(cat)/Km}{k(uncat)}[/math] and [math]efficiency = \frac{k(cat)}{Km}[/math] respectively. Can anyone shed some light on this for me? Cheers Kaeroll

The only ones I've thought of are using A535, fueling brain cells and the consumption of medicine. Anyone have any other applications?

hello! im new with this site. I just want to ask what is other alternative way of calculating the number of ATP produced in oxidative phosphorylation in glucose molecules? Hope you can help me with this. Thanks a lot.

Could someone explain to me how the total number of molecules of water produced for the complete oxidation of palmitoyl coA to water and CO2 is calculated?

Can someone give me a link to an interactive exercise to identify amino acids?

Hi All, Does anybody know off the top of their head what the maximum size of DNA that can be successfully electroporated is? I have been looking all over, and have not yet found a bp size that one should not go over when trying electroporation. Is it on the order of billions, millions, or thousands of base pairs? Thank you in advance, Chris M

I know that these serve to transfer electrons from cytosolic NADH to the respiratory chain. I also know that the malate shuttle generates NADH in the mitochondrion, while the glycerol phosphate shuttle generates FADH2, meaning that cells that use the malate shuttle will get more ATP from a molecule of glucose than those that use the glycerol phosphate shuttle. What nobody's been able to explain to me, though, is why any cells use the glycerol phosphate shuttle at all. (And yes, I know that different cells in the same organism use different shuttles). Is it a problem with enzyme regulation perhaps?

Hey Does anyone know of an in vitro in vivo correlation for the DNAse activity? How relevant is an in vitro digestion using 5000 Kunitz units to what is found in a biological milieu (cell or whole animal)?

Hello there, We are using the Roche TeloTAGGG Telomere Length Assay to measure telomere lenth in peripheral blood mononucleocytes. (manual is found at https://www.roche-applied-science.com/pack-insert/2209136a.pdf). It is a non-radioactive chemiluminescent assay, that involves digestions, gel electrophoresis, telomere probing... We have been having a very weird problem. Our film shows nice smears for each lane, but it is like a small river flowed across the lanes. No one has a clue what would cause it, and it has been very frusterating given the cost and time we have used already. Here is a link to a picture of our film. Does anyone have any clue…

I'm getting very low amounts of RNA when extracting it with TRIzol form under culture differenciated human dendritic cells. I usually don't get more than half a microgram. Could somebody help me or has have the same experience?

When using a P-200 and a P-1000 Rainin Pipetmen which one is more accurate and which one is more precise? Why?

Dear people, I have this question on whether is it possible to clone these 3 genes which has the antibacterial, antifungal and antivirus activities into a vector and expresses them? I have tried to find journals related to this, but to no avail. Hope you guys can help out. Please let me know if it's possible, what are the requirements and how can it be done. It would be better if you can attach the links from where you obtain the information from. Thanks. Cheers!

HI, I was just wondering if it was ok to store your PCR in -4C overnight and thawing to do a PCR clean up the next day? Will this screw up my results or should it be ok to do THanks, Christy.

Hello, I'm trying to do some biophysics - but I suck at it! I have two beta-sheets and I'm trying to work out how they'll 'connect' together, i.e. which functional groups will associate. I know that hydrophobic ones (e.g. Ile) will cluster together. I also know that aromatic ones (i.e. Phe) might stack in pi-bonding. But could anyone explain to me in simple terms which other amino acids would 'like' to be near each other? I'm not sure about the positive or negative charged ones, or the polar ones. Thanks!!

I read an abstract which mentioned "amino peptide", could anyone tell me what does the "amino peptide" mean in that article(I can not read Russian)?? I am puzzled:Is there any peptide with only N-end or C-end？？ Cultivation of Haemophilus influenzae, serotype B, in amino peptide-based semisynthetic nutrient medium] [Article in Russian] Orlova OE, Vaneeva NP, L'vov VL, Iastrebova NE, Elkina SI, Sergeev VV, Kalina NG, Zakharova NE. Mechnikov Research Institute for Vaccines and Sera, State Research Center-Institute of Immunology, Moscow, Russia. The work shows the possibility of the cultivation of H. influenzae, serotype b, in semisynthetic nutrient medium …

which enzymes is used for replication:-(

I have taken an oral exam in TURKEY to qualify as an Assoc. Prof. The following question was asked and I did not know the answer, I failed the exam. They will ask it again. Pregnant women carries table sugar (sucrose) and when she gets hungry, she takes sugar, thus ketone bodies production decreases. If she is hungry for a long time, ketone body production will increase and that will cause Diabetes. In other words, Ketone bodies production causes Diabetes, what is the mechanism behind this phenomenon? As I know, Diabetes patients have higher ketone bodies production, since they can not use Glucose, body breaks down fats and free acids to Acetyl CoA which for…