BabcockHall

I don't see dilute HCl in your protocol. Dilute is a little bit vague, but remember to add commercial HCl to water to prepare it, not the other way around.

I was not able to follow everything you said. However, I have some general suggestions. Sometimes a sample is diluted as part of its being assayed. Occasionally, a sample is so concentrated, that it has to be diluted before a portion is taken for an assay. I would first calculate the concentration of the analyte in the assay, then I would do each dilution calculation separately, assuming that there were two dilutions. The amount of substance that one possesses is usually only of interest for the initial solution. That value is found simply by multiplying the concentration of the initial solution by its volume.

If DCM encountered a non polar molecule such as hexane, they could interact via dipole, induced-dipole forces.

I agree that DCM has a permanent dipole moment. Water obviously has a dipole moment. What about caffeine? If it has a dipole moment, then that would produce dipole-dipole interactions in either case.

All molecules undergo London forces, but they are not always the dominant interaction. Does dichloromethane have a dipole moment? Besides H-bonding, are there other interactions between water and caffeine?

The free antibody and the the free antigen can combine to form a complex: Antibody + antigen <==> antibody•antigen The larger the association constant, the greater the probability of finding the antibody and antigen together as a complex, as opposed to each one being a free chemical species. The association constant measures how strongly the antibody and antigen are bound together. The double-headed arrow in the equation above is intended to convey the fact that this process is reversible.

The symbol pK in this context is the same thing as pKa. I am not sure what you mean by acid value and conjugate base value. What do you know about buffers?

Assuming that the cDNA of interest does not already have restriction endonuclease sites, do you have any thoughts on how you can add them?

Why don't you give your thinking on this problem? I would start by writing the balanced equation. Here is one hint: I often find it helpful to assign oxidation numbers to carbon atoms. The oxidation number of any carbon atom may be defined as the number of bonds to oxygen or nitrogen minus the number of bonds to hydrogen.

It seems to me that you are asking two very different questions. The way a chemist might define affinity (probably by the magnitude of the association constant) is independent of the concentration of the antigen. However, in your second sentence you are discussing the amount of the antibody that is produced, which is something different. On the latter question I don't know enough about immunochemistry to offer a strong opinion, but I am a skeptical of the possible claim that a smaller amount of antigen would produce a greater amount of antibody.

There are a couple of books that are designed to help students who are taking their first biochemistry course. One is called "PDQ Biochemistry" and the other is called "Basic concepts in biochemistry." I think that they are both worth a look, but I am not 100% certain that they are what you want.

My understanding of Felix Carroll's table of gas-phase acidities (Table 7.4, p. 406) suggests to me that benzoic acid is actually stronger than formic acid in the gas phase.

Butanol and water are not miscible, although ethanol and water obviously are. When we consume ethanol, its concentration is quite low in our bloodstream, around 0.1%. BTW different countries calculate BAC slightly differently, but I think that the difference is small enough to be neglected for the purpose of this conversation.

I might offer a guess that it has something to do with solvation. How could you test that hypothesis?

Start by looking both compounds up in a table of pKa values.

Hello Everyone, http://www.gelifesciences.com/file_source/GELS/Service%20and%20Support/Documents%20and%20Downloads/Handbooks/pdfs/GST_gene_fusion_system_handbook.pdf We are trying to understand a plasmid we were sent, and my knowledge of cloning is out of date. The plasmid consists of the phosphatase of interest (of known sequence) cloned into a vector designed to make GST fusion proteins, namely pGEX-4T-1, and there is a site for thrombin cleavage. After the Arg-Gly thrombin site are six nucleotides that would express a serene and a proline residue. The next nucleotides begin a restriction site for EcoRI, GAATTC. The paper we are following indicates that EcoRI and XhoI were the enzymes used to clone the gene of interest. "Collectively, the pGEX vectors provide all three translational reading frames beginning with the EcoRI restriction site (Fig 1.1). pGEX-1λT, pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from λgt11 libraries." Based upon this passage from the handbook above, I would offer a guess that the phosphatase gene was cloned out of lambda-gt11, but I don't know this with certainty. What I don't know is what lies between the EcoRI site and the beginning of the gene for the phosphatase. Is there any way to make an intelligent guess about this? is there any resource that describes cloning from lambda-gt11 into pGEX-4T-1? We have the mass of the fusion protein, and we would like to compare the predicted versus the theoretical mass to verify the existence of the thrombin cleavage site. Thanks for any help or suggestions.

You obtained the correct number of high-energy bonds, but you used nonstandard accounting. The bond between the alpha and beta phosphorus atoms in ATP is a high energy bond, so counting that one is fine. The hydrolysis of pyrophosphate to 2 molecules of inorganic phosphate is the other bond that is spent. Putting it another way, the sum of the reactions of an aminoacyl-tRNA synthetase and inorganic pyrophosphatase creates a charged tRNA but it also turns ATP into AMP and two molecules of inorganic phosphate. To resynthesize ATP requires that two phosphoanhydride bonds are re-formed.

I have been ignoring initiation and concentrating on adding one amino acid residue to a growing polypeptide. My bookkeeping is a bit different. There is no energy input in the step (sometimes called transpeptidation) in which the new peptide bond is actually formed. I agree that there is one high-energy bond spent for recognition and one for translocation. How many high-energy bonds are spent to make a charged tRNA?

Does anyone know whether thrombin that is inactivated by PMSF would bind to a benzamidine affinity column? I can argue it either way.

I teach a biochemistry laboratory course, and we use various protein shakes as our unknowns. I prepare them at 5 mg of solid per milliliter in DI water. I gravity filter them, and next I filter them through 0.45 micron syringe filters to remove suspended matter. These two steps are very slow, and the 0.45 micron filters have to be changed several times (possibly centrifugation would clarify them adequately). However, the resulting solutions generally behave well in Bradford assays. Sugars should not interfere with a Bradford assay, but they could interfere with other colorimetric assays. Some vitamins absorb in the UV region as well, and these might also interfere with a UV assay.

What are 2-FMA and 4-FA? Without names or structures, one lacks a critical piece of information. Also, It is unclear to me what you are asking? What question does this test address? Are you having difficulty performing it?

As a general rule discussion boards like this don't dump answers to questions. The participants help the OP help himself or herself. I suggest that you Google "Williams pKa table" if you don't already have the values. If you do, what is the structural difference between the two? I am not sure what you mean about the second pKa values.

Why don't you attempt to explain it first? Then we can comment on it. A good place to start is to give the pKa values and indicate the structural difference between the two.

We are making a GST-fusion protein for the first time. Typically these are eluted from an affinity column with 10 mM glutathione: From an article on the subject, "10 mM glutathione buffer: 50 mM Tris, 10 mM reduced glutathione, pH 8.0 (make fresh daily)" http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584333/ For some applications the next step is removal of GST tag by cleavage with a protease (thrombin in our case). Thrombin contains several disulfide bonds. Some years ago, I looked into the issue of reducing agents and how they might inactivate thrombin. My recollection is that low concentrations of certain biochemical reducing agents could be tolerated, but high concentrations could not be. What I am having a hard time learning is whether or not 10 mM glutathione will reduce and inactivate thrombin. On the one hand, 10 mM is a decent concentration; on the other hand, glutathione might be too bulky to react with thrombin quickly. Ordinarlly I would just do a buffer exchange, but we are trying to minimize the number of steps of things like buffer exchanges, because our protein can drop out of solution under conditions that we have not been able to predict or control yet.

HYDERABAD: In an alarming trend, an increasing number of women from across the city who wear the burqa or observe purdah are being diagnosed with vitamin D deficiency on account of inadequate exposure to sunlight. Exposure of at least hands or feet to sunlight every day for 15-20 minutes is necessary for the vital compound to be absorbed into the body, advice doctors. "Around 15 per cent of the body should be exposed to sunlight for around 20 minutes a day. It could be hands or feet," National Institute of Nutrition deputy director Dr N Lakshmaiah told TOI. "Otherwise supplements in the form of conventional tablets should be used regularly." link When I was a graduate student, Hector DeLuca told us about a religious sect in which veiled women were suffering from this deficiency. The leader(s) then set up a kind of ritual, where the women left the men en masse for a few minutes each day and removed one or more veils to allow for production of vitamin D.