Protein identification and analysis software performs a central role in the investigation of proteins from two-dimensional (2-D) gels and mass spectrometry. For protein identification, the user matches certain empirically acquired information against a protein database to define a protein as already known or as novel. For protein analysis, information in protein databases can be used to predict certain properties about a protein, which can be useful for its empirical investigation. The two processes are thus complementary. Although there are numerous programs available for those applications, we have developed a set of original tools with a few main goals in mind. Specifically, these are: 1. To utilize the extensive annotation available in the Swiss-Prot database wherever possible, in particular the position-specific annotation in the Swiss-Prot feature tables to take into account posttranslational modifications and protein processing. 2. To develop tools specifically, but not exclusively, applicable to proteins prepared by two dimensional gel electrophoresis and peptide mass fingerprinting experiments. 3. To make all tools available on the World-Wide Web (WWW), and freely usable by the scientific community. In this chapter we give details about protein identification and analysis software that is available through the ExPASy World Wide Web server.

Protein identification and analysis tools in the ExPASy server

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

/pdf/loop-mediated-isothermal-amplification-of-dna-3hidm8kki0.pdf

Loop-mediated isothermal amplification of DNA

The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:3193261 and is based on data from Edelhoch [1967, Biochemistry 6:1948-19541.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, 6 M guanidine hydrochloride (GdnHCI), and 8 M urea have been measured. For Trp, the average E values for the proteins are less than the E values measured in any of the solvents. For Tyr, the average E values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured t values for 80 proteins, the t at 280 nm of a folded protein in water, t(280), can best be predicted with this equation:

https://onlinelibrary.wiley.com/doi/pdf/10.1002/pro.5560041120

How to measure and predict the molar absorption coefficient of a protein.

http://ctrstbio.org.uic.edu/manuals/kellybba.pdf

How to study proteins by circular dichroism

Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.

Full-length RNA-seq from single cells using Smart-seq2

Calculation of protein extinction coefficients from amino acid sequence data

http://galton.uchicago.edu/~lalley/Courses/312/Berg1989.pdf

Facilitated target location in biological systems

We show that the amino acid analogue betaine shares with small tetraalkylammonium ions [Melchior, W. B., Jr., & von Hippel, P. H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 298-302] the ability to reduce or even eliminate the base pair composition dependence of DNA thermal melting transitions. The "isostabilizing" concentration of betaine (at which AT and GC base pairs are equally stable) is approximately 5.2 M. Betaine exerts its isostabilizing effect without appreciably altering the conformation of double-stranded DNA from the B form. The presence of > 5 M betaine also does not greatly change the behavior of DNA as a polyelectrolyte; this lack of effect on electrostatic interactions is expected because betaine exists as a zwitterion near neutral pH. Study of DNA melting transitions in high concentrations of betaine thus allows the experimental separation of compositional and polyelectrolyte effects on DNA melting. As a consequence, betaine solutions can also be used to investigate DNA-protein interactions under isostabilizing (or close to isostabilizing) conditions, which has not been possible using isostabilizing salts. This potential is illustrated by examining the highly salt concentration-dependent interaction of ribonuclease A with DNA in concentrated betaine solutions.

Betaine can eliminate the base pair composition dependence of DNA melting

In this paper we summarize the various factors that must be considered in establishing the operational specificity of the binding of a protein regulator of gene expression to a DNA target site. We consider informational (combinatorial) aspects of binding-site specification, actual recognition mechanisms, and the thermodynamics of target-site selection against a background of competing pseudospecific and non-(sequence)-specific DNA binding sites. The results provide insight into the design, specification, and possibly the evolution of regulatory proteins and their chromosomal binding targets, as well as into practical aspects of the design of regulatory-protein isolation schemes and physicochemical regulatory considerations in vivo.

https://www.pnas.org/content/pnas/83/6/1608.full.pdf

On the specificity of DNA-protein interactions.

Pre-steady state kinetics of misincorporation were used to investigate the addition of single nucleotides to nascent RNA by Escherichia coli RNA polymerase during transcription elongation. The results were fit with a branched kinetic mechanism that permits conformational switching, at each template position, between an activated and an unactivated enzyme complex, both of which can bind nucleotide triphosphates (NTPs) from solution. The complex exists most often in the long-lived activated state, and only becomes unactivated when transcription is slowed. This model permits multiple levels of nucleotide discrimination in transcription, since the complex can be "kinetically trapped" in the unactivated state in the absence of the correct NTP or if the 3' terminal residue is incorrectly matched. The transcription cleavage factor GreA (or an activity enhanced by GreA) increased the fidelity of transcription by preferential cleavage of transcripts containing misincorporated residues in the unactivated state of the elongation complex. This cleavage mechanism by GreA may prevent the formation of "dead-end" transcription complexes in vivo.

https://science.sciencemag.org/content/sci/262/5135/867.full.pdf

P H von Hippel

Papers

Calculation of protein extinction coefficients from amino acid sequence data

Facilitated target location in biological systems

Betaine can eliminate the base pair composition dependence of DNA melting

On the specificity of DNA-protein interactions.

Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription.