Biochemistry and Molecular Biology

Hello, is it possible for large molecules of proteins - hydrolases (10 - 50 kDa) to be absorbed (in their -fully functional form) through the wall of small intestine to blood? Enzymes got to the intestine because it was protected against digestion in stomach by medicament cover. If yeas, is it possible through the mechanisms of persorption by slubs, tight junctions or by random lymphocytes carrying enzymes? Thank you for your answers!

A student whom I know wishes to do some DNA melting experiments, following them by UV/VIS spectrophotometry. What sort of solvent should she use for a recirculating bath? I have used ethylene glycol/water for low temperature work, but I have not done much high temperature work. If anyone knew of a Methods article on this subject, please feel free to pass along the reference.

Hello, I am wondering what I could expect when doing a yeast two hybrid experiment with 96 spots. When I run my experiment (1 bait vs 96 preys) I often end up with lots of growth on my final selection plate (the one you use to check for interaction). I often have 15 spots that show growth. Now I plate them out twice (I am doing it manually, so I just make 2 selection plates for every test): so on 2 plates to select for interaction. The weird thing is that the growth often does not match. I see growth on different spots. Often I have, eg. 15 spots that grow on plate 1 and 17 spots on plate 2 and 10 of them will match for example. (and I already quit incubating …

Hi all, I'm wondering why there are more scientific papers published on methylation over acetylation. On PubMed, there are 50k results when I type in "DNA methylation", but only 12k results when I type in "histone acetylation". Is there any clear reason why? Methylation being easier to assay, having more clinical importance, or being discovered earlier? Please let me know .

I'm a little confused about one step regarding glycolysis and the glycerol phosphate shuttle, I'm going to walk you through my thoughts.. * During glycolysis we get 2 NADH for every glucose molecule * NADH needs a shuttle system to pass the inner membrane of the mitochondria * The first step of the glycerol phosphate shuttle is when the GPDH-C enzyme catalyzes the following redox reaction: DHAP + NADH -> 3PG + NAD+ * As I see it we are getting down on lower energy state regarding both 3PG and NAD+. NAD+ is obviously at a lower energy sate as NADH is carrying energy in form of electrons. Looking at the glycolysis process we gain 1 NADH and 1 ATP by going from …

Good Afternoon, We would like to remove thrombin from a post-nickel column digest. However, our protein and thrombin have similar molecular weights; therefore, I don't hold out much hope for gel filtration. We think that cation exchange may work, inasmuch as our protein has a low pI. We are interested in co-crystallization experiments with a second protein. There is a benzamidine column, but it is a little pricey. There is also biotin-labeled thrombin, but it has the same drawback. There is also APMSF as an alternative to PMSF, and this would at least inactivate the thrombin. Thanks for any suggestions.

Hey, so, I have a class on Health Education and got assigned to do a demonstration on a topic from Biochemistry. I chose the sub-topic of how nonessential amino acids are synthesized and have to help my fellow students: Define what biosynthesis is Outline the reactions and intermediates The mechanisms for each reaction Explain how not having any of the nonessential a.a.'s is not entirely unhealthy I'm also making a table containing the nonessential amino acids along with what reaction synthesizes it, it's substrate, and intermediate. I'm stuck at trying to figure out which parts of an illustrated reaction is part of the substrates. I'm also not sure i…

Hi all, Desperately need help here.. I am currently trying to anneal two complementary single stranded oligos. They are about 44bp long and the tm is about 57C. I initially just heated the oligos in annealing buffer (10mM Tris-HCL pH8 + 1mM EDTA + 50 mM NaCL) to about 98 degrees and let it cool to room temp gradually. But when i ran them on TBE gel (4-12% gradient) i found that alot of it are still forming single strand. These oligos are AT rich so i assumed the hairpin structure is the problem, although when i ran it through oligoanalyzer program the energy is still highly favourable for double stranded annealing rather than self dimer. I since tried varying diff…

This is the first time I do WB with p-STAT1 or p-STAT6 for inflammation. p-STAT1 (tyr701) and p-STAT6(Tyr641) and both are from Santa Cluz Biotechnology. Treatment of LPS+IFN-y for expression of p-STAT1 , and IL-4 for p-STAT6. I try to WB many time but it still has no band. Is there anyone having this experiment can give me the conditions for WB these protein? Thank you very much!

I support GMO's. I believe they're safe and I have not found any groundbreaking research that states otherwise. -I haven't read any papers or articles on specifically why David Suzuki and also Bill Nye oppose GMO's. I haven't heard them reference ant reputable journals to support their argument. ~ee

Hi all, I'm using SensoLyte® 520 HIV Protease Assay Kit *Fluorimetric* of Anaspec to determine the activity of my HIV-1 protease. I have some problems in calculating Kcat value. I have calculated Vmax and Km follow Lineweaver-Burk plot are 6.97 RFU/sec and 3.7 uM, respectively. These two values are roughly equivalent to the parameters that Anaspec launched. But the kcat value is many smaller than Anaspec's value. My enzyme weight is 24500. I used 200 ng enzyme/1reaction with a total volume of 30 uL. And Vmax was converted to nM using a produce standard curve in an attachment file. Please give me some suggestion to determine kcat. Thank you

Dear all, I would like to know if it is possible to derive kon k_on and koff rates from Michaelis-Menten constant? The reason is I have MM constant from experiments, but I actually need for kon k_on koff to build a mathematical model in terms of mass action. Thank you all!

Hey Guys I'm doing a little research into lipid metabolism and uses in industry but I can't seem to have any luck with caprylic acid. I know this lipid is produced industrially and found in small traces in coconut oil and mammalian milk but I can't find anything on HOW its produced industrially nor can I even find a metabolic pathway . Any of you budding scientists know any articals/books or journals that may help my search or better yet can anyone offer me the knowledge?

Why is it not possible to upregulate the expression and activity of sex steroid enzymes?

Pasteur's swan-neck flasks demonstrated that microbes are air-borne and do not "spontaneously generate" within flasks... even with an air supply. Utilizing gravity only, his S-shaped swan-necks kept out microbes remarkably well. For sprouting, a breathable fabric and regular water changes are advised, but even ad hoc seed sprouters don't utilize gravity from what I can tell. Why don't they utilize gravity? Just boil the water beforehand and you won't need to change it, right?

I am having trouble understanding why and when the mitochondria generate ATP. I am trying to explain it without using technical terms such as chemiosmosis etc. Here is what I understood - please correct me if I am wrong. Also, for each point (in bold), I have a few questions. Mitochondria breaks glucose into ATP. Does this happen as soon as we eat? What about the rest of the time when the food has been digested? Does the glucose come from breakdown of glycogen, or is that only for the muscles? Is ATP ever made from scratch? Mitochondria breaks 1 glucose molecule in a series of steps during cell respiration. The by-products (NADH, FADH) form a total of 38 ATP. If…

Hello, everyone! Could you please answer on some questions. I want to know about a bilirubin-albumin bond and penetration of blood brain barrier(BBB) of the healthy adult(not infant). I have some questions: 1) I read that unconjugated bilirubin is bound with albumin don't penetrate in tissue. Then why does human become yellow with elevated levels of bilirubin(eg 5 mg/dL, albumin can bind 20-25 mg/dL) and normal level of albumin? 2) In the books written that unconjugated bilirubin even with severe and long-standing jaundice can't penetrate the adult brain. Then why human who has >2-3 mg/dL of bilirubin feels neurological symptoms? 3) Can unbound unconjugated bilirub…

I heated a piece of banana peel in conc. Hcl and gradually the solution developed a blood red color !!!! Any speculations over what may have caused this ???

Hi everyone In preparation for my Molecular Biology & Genetics exam, following example question: "Which of the following proteins polymerizes nucleotides in DNA?" With 2 of the possible answers being "DNA-ligase" and "reverse transcriptase". Intuitionally, I'd go for the last one. However, discussion arises with the question if it polymerizes "in" DNA, considering the transcription of RNA to DNA. The big question of which the answer might solve the issue, is the following: can a ligation be put equal to a polymerisation? As in, if 2 or more nucleotides are bound, can one say that they are polymerized? (Which is the case for okazaki frags.) Thanks in …

I'm looking for a way to modify mitochondria to more efficiently produces ATP. Right now i'm considering increasing COX production. Even though it causes cell death. http://www.sciencedirect.com/science/article/pii/S0005272811001605 My other idea was to find a more efficient eukaryote and 'swap out' whatever prekaryote out cells merged with long ago. Anyone know of a eukaryote with a higher efficiency rate than 38% Thanks in advance.

Hi everyone In preparation for my exam molecular biology, this question was asked: When a cDNA-fragment of a eukaryotic gene is built in in a vector on a position after the lacZ translation-initiation place with EcoR1-linkers, the chance of getting an in-frame coupling would then be: A. 1/2 B. 1/3 C. 1/6 D. Very small And the answer would be C. However, I can't seem to understand why exactly. Could someone help me on this one? I'd manage to find out already 1/3 (GAATTC can be built in in 3 ways, of which 1 won't cause a frameshift). Thanks! F.

Hello again, I have extracted RNA and i am satisfied with the purity (260/280 ratio) and the yield. What is troubling me is the very low 260/230 ratio? Therefore, i wanted to know how important is the 260/230 ratio in RNA extraction. I will use the RNA for qPCR. Yield 26-35 ng/ul and 260/280 ratio is between 1.9-2.2 If i want to re-precipitate the RNA should i use sodium acetate or Lithium chloride to do this? Thanks

Hello, I want to understand how do i analyze melt curve after my qPCR? What are the different curves? I am just aware that melt curve analysis is done to check for secondary products or primer dimer formation. I would appreciate your help in understanding the melt curve. Thanks

Hi, I got a PCR efficiency for all my targets and samples as 1. I did a quantitation analysis but it still calculated it as 1. I know that the R^2 value should be 0.9933 and slope should be -3.32 then by formula we get 1. But as i didn't run a standard curve, how reliable is this efficiency result? (lot of questions today). Thanks

Hi, I know this question must have been answered before but i would like to confirm it again. I am using Thermo-scientific cDNA synthesis kit and it says take 1pg - 5ug of your template for cDNA synthesis. I am having a RNA yield of 19 ng/ul (micro-dissected) brain sample i.e. the hippocampus with a total volume of 50 ul. I then treat it for DNAse. So, my total volume is not more than 55 ng/ul and i want to save some RNA for future experiments. Now, to calculate 1 ug, x ul = (1 ug)/(25ng/ul) x ul = 1000/19 x = 52.63 ul Until now i was using about 2 ul of the template to synthesis cDNA. Will it be fine with 2 ul or should i inc. the volume to 50 ul for c…