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11

The short summary is that typical TFs bind and read both strands together, as a basepair sequence. Some proteins instead recognise a site on the helix by its shape and flexibility. ssDNA-binding proteins obviously bind one strand but they do this in a non-specific manner. RNA-binding proteins recognise the sequence on a single strand by inserting ...


9

The techniques used to do this are ChIP-seq and ChIP-chip. Basically, you let the pathogen bind to the (highly replicated) DNA cut up the DNA into little random pieces by sonication enrich (“pull down”) the pathogen-bound DNA fragments by using a known antibody which binds to the pathogen sequence the thus enriched DNA map the sequenced fragments back to ...


7

I found article in Nature: A helper phage to improve single-chain antibody presentation in phage display Experimental protocol Standard cloning procedures, determination of colony-forming units and plaque-forming units, and immunoblot after PAGE were carried out according to Sambrook et al. Construction of the packaging cell line. ...


7

16 units/mg means 16 units per milligram of protein. Many companies, including Invitrogen, define 1 unit streptavidin as the amount of streptavidin necessary to bind 1 microgram of biotin.


7

See this paper. They have studied RBP-protected sites in the entire human transcriptome by RNA-protein crosslinking followed by RNAse digestion and sequencing: PIPseq. Figure 1 of the paper shows distribution of protein protected sites in RNAs. They also correlate it with different regions of mRNA and its expression. They show number of protein protected ...


6

in silico modelling of anything in biology is an active field of research. It's very useful for making predictions and developing hypotheses, but any findings need to be confirmed experimentally. From the Folding@Home website: Folding@home has been a success. In 2000-2001, we folded several small and fast folding proteins with experimental validation ...


5

Just to add to Chris Stronk's answer: 1 U SAV can bind 1 ug biotin This tells you that in a 16 U/mg SAV sample, every mg of SAV will bind 16 ug of biotin. You can figure out the molar ratio from this: $16\mu g\ BIO\cdot\frac{1mol\ BIO}{244310000ug\ BIO}\cdot\frac{52800000mg\ SAV}{mol\ SAV}$ Which equals: $\frac{3.46mol\ BIO}{mol\ SAV}$ Theoretically, ...


5

This is a classical protein purification problem - you have to find ways to fractionate your mixture so that each fraction can be assayed for the activity you are interested in. When you find the active fraction you then subject that to a different type of fractionation. Salting out The solubility of proteins is affected by the ionic strength of the ...


5

See here. Histones are basic proteins (cationic, high pI) because they are required to interact with polyanionic DNA at physiological pH. Heparin and dextran are polyanions which form insoluble salts with the cationic histones.(Dextran is a polymer of glucose. In dextran sulphate it is derivatised with sulphonate groups creating a polyanionic material.) ...


5

This is the figure the question is about. On the right is the control experiment with GTP-γS, on the left without it: The bands that are visible in both experiments are unspecific binding. If GTP-γS doesn't affect their presence, the mechanism by which they bind to the column can't be specific to the GTPase functionality. The proteins the authors were ...


4

I don't know of any examples of this but I would say no doubt, that's quarternary structure. Quarternary isn't so much defined by the kind of interaction but much more the fact that it's between different polypeptides; all lower-level structures are within one polypeptide. (Wikipedia agrees.)


4

ChIP-exo does seem to be the "ChIP-seq killer." I've seen Dr. Pugh present it a few times, and the audience is pretty much always impressed. One thing I'd do if I were of the "experimental bent" would be to add random degenerate barcodes in the library prep to control for potential PCR artifacts. I imagine that since the "peaks" in ChIP-exo seem to be quite ...


3

1) Is the attachment of zinc regarded as a type of post-translational modification? It is not really considered a post-translational modification because the zinc atom is not covalently bound to the protein. Binding to zinc is adsorption. 2) When carbonic anhydrase is denatured, is the zinc ion released in the medium? Yes, but it depends on ...


3

Telomerases are tightly controlled on all level: Expression, post-translational modifications and by external factors. I think for this the external factors, a protein class called telomeric proteins. They bind to the end of the telomere and regulate the telomerase. The figure is from this paper, which looks into the topic quite extensively: Regulation ...


3

The short answer is that the Edman degradation is a multi-step process. The Wikipedia page has a decent picture of the mechanism. In practice, the peptide is reacted with phenylisothiocyanate (PTH) under mildly basic conditions to give a thiourea, which is stable. The excess PTH is separated from the thiourea intermediate. The thiourea is then treated with ...


2

Like any other protein, antibodies will aspecifically bind nitrocellulose or PVDF membranes, but any other protein present in your antibody preparation will also do. Depending on the antibody class, more specific binding can be obtained with protein A or protein G, that recognize the Fc domain. It's usual to have protein A/G immobilized on a stationary ...


2

In prokaryotes the glucose transporter is always present in the cell membrane; in cells whose glucose uptake is insulin-regulated the transporter is only present in the plama membrane when hormone levels are high. GLUT4 is the isulin-regulated glucose transporter found in muscle and adipose tissue. When insulin levels are low the GLUT4 protein is in the ...


2

Background binding in this case would be the extent to which two proteins associate together by chance. A hypothetical example: you may have a mitochondrial protein import complex. Usually cases there is a specific peptide sequence the import complex binds to , but proteins without an import peptide sequence will occasionally be bound to the import ...


2

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2866014/ In this work they find that formaldehyde crosslinking happens by formation of a methylol adduct (due to nucleophilic attack by N or S in case of proteins) in protein which then attacks the DNA or vice-versa. The final crosslink is by a methylene bridge Formaldehyde can react to amino groups in ...


2

I've always just gone with the name hetereodimer but there isn't any technical reason why it isn't a quaternary structure. As for an example, I work with antibody fragments or FAbs and cys-diabodies which are exactly that, two distinct polypeptides that are connected by a disulfide bond linkage. These do have a significant amount of non-covalent ...


2

A colleague of mine discovered the cipher that determines TAL effector DNA specificities, which is described in this short paper. These specificities were determined by observing TAL effectors bound to DNA and recording how often a given repeat-variable diresidue (RVD) would correspond to a given nucleotide (using a weight matrix). Now that the ...


2

The following paper answers your question in detail: http://www.ncbi.nlm.nih.gov/pubmed/18568020 It seems like the following aa are responsible for the binding, but please refer to the paper for more details: Val152,Tyr156,Asn157,Ser422,(Asp79)


2

Take a look at the paper by Ojasoo et al. [1] It reports the relative binding affinity for a wide variety of steroids for the AR, PR and GR. Nandrolone (19-nor testosterone) does not have any affinity for the GR, but is a potent activator of the AR. However, it does have some affinity for the progesterone receptor (does this matter for your research?). ...


2

A second telomerase is not necessary, as the lagging strand is filled in by the DNA-Polymerase, see this figure:


2

The sRNA mediated regulation in bacteria operates via diverse mechanisms. This case of sRNA competing with a mRNA for a protein is a passive kind of regulation. This might be good for finetuning but may not be very efficient. It is much better to actively regulate a mRNA by direct binding. Also, it will work only if the concerned protein is in limiting ...


2

Addition to the answer provided by Teige. Transcription factors bind to both the strands however your question also included proteins in general. DNA remains double stranded and twisted as helix most of the times; most proteins bind to both the strands as mentioned in the previous answer. However some proteins such as SSB (Single-strand binding protein) ...


1

I will try answer your question directly. How do we know if we fold it right? A. If you're interested in only the end product pf folding -- the 3D structure, then this is the subset of the folding problem called the structure prediction (from sequence alone). a. We can verify the structure experimentally by determining the 3D structures by NMR or ...


1

A certain fold of a peptide string can be validated or ruled out if other experimental data is available. Some other techniques to infer protein structure are X-ray crystallography (requires pure protein that will crystallize) and single particle analysis.


1

Katsamba PS, Park S, Laird-Offringa IA. 2002. Kinetic studies of RNA–protein interactions using surface plasmon resonance. Methods 26(2):95-104 There are many methods for studying kinetics, I only mention this one because the lab I'm in has used it for studying protein-ligand interactions. That article has a good explanation of SPR and how it works, so I ...


1

Ridgeway and coworkers designed a really neat microfluidic device for making these kinds of kinetic binding measurements on RNA and protein (Ridgeway et al. 2009). Their device works sort of like a stop-flow, and they used a FRET based scheme to detect binding and unbinding events. They took measurements for one particular rRNA/ribosomal protein pair and got ...



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