Biochemistry and Molecular Biology

I've been having issues with my anion exchange chromatography column on an FPLC that I was hoping someone would be able to help with. The column is a GE ResourceQ 1 mL. I'm using the column as a first step in purification of an enzyme, I've previously done the chromatography (two months ago) with very good results. I was just barely saturating the column with enough input material to generate a main peak of around 1400 mAU (so binding wasn't an issue originally). Since then, I noticed the capacity had decreased, and I was losing a lot of protein in flowthrough and only retaining enough to generate a peak of around 500 mAU. The buffers are the same formula but different ba…

Hi DH10B is host of my plasmid , but we dont have DH10B in lab. can I use top10 or DH5a instead of DH10B? (base of my plasmid is pbluescript KS+ )

Hello everyone, I have a question about drunk, i wonder why drunk people like to eat something is sour like young mango, lemon,...? Because i live in Vietnam and i see many drunk do that. Is that problem related to biochem metabolism in our body ? I need to know the reason, i think it is related to glucose metabolism? ( i just guess ^^). Please help me, thanks so much!

In hybridoma technique ..why do v need to kill so many mice ,cant v think of any other way in which v can obtain products without harming those animals ? Cant v use humans for this ,like instead of inducing cancer in those animals cant v take cancer cells from an affected person n lymphocytes from the spleen of a dead person who had the infection ??is there any problems if v use humans for this ?(except ethical issues)i just thought of it coz if v can use mice for this then i guess v wont have much problems with human cells!sorry if i am wrong ! I just posted what i think !(i dont know much about this technique,)

I was going to get a microsope and i found this one: http://www.microscope.com/omano-om118m4-1000x-compound-monocular-microscope-p-708.html I was wondering if this was good or if someone could suggest one.

hi!is there a mild stripping buffer for cerebellar substrates for western blot?

Hi, I have recently designed a set of primers to amplify a fragment of a gene present in an cDNA template. I have performed a blast search on my primers and the results have shown a hit with my gene of interest. However, after conducing the PCR and cloning my fragment into a vector, i sent it off for sequencing. The results were surprising. Sequence analysis showed that the amplified fragment was from a completely unrelated gene. I was wondering, bearing in mind my designed primers are specific for my fragment of interest, why I amplified a non-specific fragment. Any suggestions would help. Thank you, Biochemistry3096

Hi, So Im trying to grow my protein in Codon + RP cells in order to use them in a pull down assay. However, theyre being very tempremental and Im struggling to get all my variants to grow consistantly. They will all grow and express my proteins fine sometimes but then not a week later, despite using the same antibiotic stocks and transformation plates. This week Im having trouble with two of them. The first just wont grow although two other constructs transformed at the same time have grown fine. The second grew fine last week but wont grow this week. It was suggested that it could be a bacteriophage infection but all my other cultures all grew fine and Ive b…

I'm trying to learn more about the Lac operon, and I am confused regarding a few thing. (sorry if this is the wrong section) IPTG is a known inducer of the lac operon, which can be used instead of allolactose. My question regarding IPTG is as follows; does IPTG lead to transcription of the Lac operon or only the removal of the lac repressor? Because as I understand it, cAMP is needed as well, in order to bind the CAP activator to the CAP-binding site, so that RNA Polymerase can be recruited to the operon. So if I had glucose and IPTG, would the operon still be transcribed, since glucose leads to decrease in cAMP concentration. The second question is regarding Glyc…

So I get that your body burns energy, but on the treadmill I have to take quite a few steps before I burn a whole calorie and I very very highly doubt that the energy contained in raising a mere cubic centimeter of water by 1 degree is enough energy to move my body 10 feet at 10 miles per hour.

Ribosomes are molecularly massive structures, composed of 3 RNA strands, cradled in a cluster of 56 proteins. Those 3 RNA strands (16S + 5S,23S), and their corresponding complimentary anti-strands (16S + 5S,23S), code for what poly-peptides, i.e. would generate what proteins, if "fed" to other ribosomes ?

Hello, noble nerds, I ask for your advice on a high-stakes problem. I'm going to be collecting a large number of saliva samples, adding a preservation liquid to them, and then transporting them by mail at room temperature. The liquid basically needs to do three things: 1) lyse the saliva cells 2) provide anti-microbial protection (gram positive, gram negative, fungal) 3) buffer the DNA for preservation. There are already some commercial vendors of such a preservation liquid. But they're extremely expensive. 15+ USD for a single sample! And I need to transport thousands, this is just beyond my budget. * So, I've stitched together the below lysi…

Hi: I remember a biochemical pathway by one of the non-pathogenic species of Clostridium bacteria. This pathway convert ethanol to butyrate [which smells like cheese] and caproate [which smells like goats]. The above being said, is it possible to make goat-cheese from wine, if it is inoculated with the aforementioned microbe? Thanks, GX

I've been reading about doing immunoprecipitaiton. My first experiment failed really bad, not sure why. Anyway, I've been told I should have been doing BCA method quantification in order to determine the protein concentration of how much protein I have in a volume of solution. Well, I've been told that after taking tissue and lysing, I should do BCA. Afterward, do IP, but then don't BCA quantify and just move onto a western blot. Uh, why would I not want to know the protein concentration of the protein that I've immunoprecipitated out? Wouldn't that be what is relevant for observing what amount I've used in the western blot analysis?

I've recently been assigned the task of making a decent antibody for amyloid precursor protein (and perhaps something else, too). As I have been reading as of late, there is considerable demand for decent antibodies, as people don't tend to like what comes from industry. First off, why? Does industry not put enough effort into making antibodies, such as generating one generation of a mouse population to produce antibodies, thus the search for better antibodies beyond that one generation does not occur? Industry would be the social structure with enough money to improve on design rather than independent academic institutions, right? Is it that industry isn't re…

How does one derive the adairs equation for a dimeric protein, because my text books are not very much comprehensive and step by step oriented.

The glucose-galactose binding protein (GGBP) has been resolved crystallographically in an OPEN and a CLOSED (and TWISTED) conformation, enabling free energy differences to be computed between them (http://www.sciencedirect.com/science/article/pii/S0006349507713097) Another similar protein, RBP (ribose binding protein) similarly exists in two known resolved forms (OPEN and CLOSED) whose crystal structures exist. Mowbray, S. & Cole, L. (1992). The 1.7 A ̊ X-ray structure of the periplasmic ribose receptor of Escherichia coli. J. Mol. Biol. 225, 155–175. Bjo ̈rkman, A. & Mowbray, S. (1998). Multiple open forms of ribose binding protein trace the path of its con…

Hi there, I was just wondering if anyone can provide me with some insight here. I am a novice molecular biologist and I am involved at looking at splice variants (alternative forms of the same gene) I am able to find mRNA for these variants but when I go to look at the protein (using an antibody that detects a region that is common in all the variants) I can't see them. I tried different percentage gels (for western) etc. Is it because the message isnt translating or something about the way the protein folds? Can anybody help me regarding proteins?

I have been studying a protein that has an LPXTG motif, so it would be expected to be sorted to the cell wall. However, it only appears in the membrane fraction when I do a western blot. Does the presence of an LPXTG motif strictly dictate cell anchoring or have proteins with LPXTG motifs been known to localise to other parts of the cell?

Hey, Over the last few weeks i've been running some experiments on protein expression which has involved running cell lysate through SDS-PAGE and then using Western blotting techniques to probe for a certain protein that we're looking for. So far I've observed over and over again a double banding of the protein on the Western blot. I've been running reduced gels, so it's not a dimerisation; besides, the molecular weight of the protein is around 30 kDa and the bands are about 5 kDa apart. It's definitely not a cross reactivity either. The current hypothesis is that there is a conjugate molecule attaching itself to some of the proteins - I have been treating the c…

Lidocain pka 7.6 Prolocain pKa 8.4 We are told that a decrease in the amount of base facilitates removal of the local anesthetics, resulting in a shorter duration of action. Which has a more rapid onset? The answer was that it was Lidocaine because more exists in its base form at body pH. However, this seems counterintuitive since both pKa's are higher than the pH. I do remember reading a rule somewhere that if pKA>pH, then the group is considered to be in its acidic form when attached to an aa, but I don't know if that rule only applies to aa and what that rule means. Could someone please clarify it?

I was making an 40% (19:1) acrylamide:bis gel with TEMED and ammonium persulfate, and as a used a syringe to make the gel, the solution in the syringe polymerized, but the remaining in the beaker and the solution already between the plates was still fluid. -How do I prevent this in the future?

Hello! I'm looking for a very special protein, I know how it should look like, but it's not easy to find: - a transmembrane protein residing exclusively in the ER - ideally Type I membrane protein with short or no lumenal part - N-terminal region self-sufficient for insertion into membrane Perhaps you can already imagine, what I'm trying to do. I need this protein to label the ER membrane with a fluorescent protein in the cytosol. (An antibody staining does not help me, then I would not post this here ) It should not interfere with any biological process at the ER membrane, that's why I don't like a functional lumenal part. When anybody knows a paper, wher…

I did an experiment where we needed to calculate the enzyme activity with an inhibitor using the absorbance. The question was, why do we calculate the absorbance only between 1-3 minutes of runtime and not any longer? Why did we not use the first minute in the calculation ? My teacher said it was related to the inhibitor

I need a small clarification regarding fatty acid synthesis in humans. To be more clear with my question, I will consume some space to explain few things. consider the following image: That above diagram shows nomenclature of fatty acids: consider the fourth one alpha linolenic acid its w3, meaning the first double bond from the last carbon is in 3rd position. 18:3 means its 18 carbon and has 3 double bonds, delta 9, 12, 15 refers to the position of double bonds from the COOH end. legend for figure 2: 1, elongase; 2, delta6 desaturase; 3, delta5 desaturase; 4, delta4 desaturase The above figure shows the interconversion of fatty acids within …