Protein Quantification and Analysis Techniques PDF

2024-11-27 22-07-01 Transcribed by TurboScribe.ai. Go Unlimited to remove this message. (0:05 - 0:28) Techniques in Protein Quantification and Analysis Proteins, also known as polypeptides, are organic compounds made up of amino acids. They are large, complex molecules that play many critical role...

2024-11-27 22-07-01 Transcribed by TurboScribe.ai. Go Unlimited to remove this message. (0:05 - 0:28) Techniques in Protein Quantification and Analysis Proteins, also known as polypeptides, are organic compounds made up of amino acids. They are large, complex molecules that play many critical roles in the body. They do most of the work in cells, and they are required for the structure, function, and regulation of the body tissues and organs. (0:28 - 0:57) They also assist with the formation of new molecules by reading the genetic information stored in DNA molecules. This topic class is about the separation, quantification, and analysis of all the proteins. There are various platforms and methods are available to quantitate the proteins. (0:57 - 1:20) Example is the spectrophotometry or spectrophotometric assays of protein in a solution. Spectrophotometry does not require either enzymes or chemical digestion or separation of the mixture prior to the analysis. We have three major types of spectrophotometric assays. (1:20 - 2:00) We have the UV absorbance method, dye binding assays using colorimetric base detection, and using defluorescent base detection. Although these assays can be run at a high throughput, they require an appropriate protein standard or constituents amino acid sequence information to make an estimate of the concentration. The choice of method used to determine the concentration of the protein or peptide in a solution depends on many factors. (2:00 - 2:31) So depending on the users and the quantity you want to extract, that is what you will choose for the assay. It can be UV through colorimetric or fluorescent base detection. The first type is the UV spectroscopy method, detecting the proteins with absorbance at 280 nanometer. (2:31 - 3:08) At 200 nanometer, amino acid residues with aromatic rings in a protein absorb the light. So the aromatic amino acid class, aromatic rings, that is amino acid that includes phenylalanine, tryptophan, or tyrosine. So class, please take note that the method depends on the relative number of aromatic rings, primarily the tryptophan and the tyrosine residues present in a protein. (3:08 - 3:30) So it depends on the number of aromatic rings present. So this method class particularly useful for detection of proteins eluting the chromatography columns as there is no loss of proteins. So the possibility of nucleic acid contamination should also be considered and compensation can be made. (3:31 - 3:56) So this method can be used to detect proteins in the 20 to 3000 microgram range. So the advantage of using absorbance at 280 nanometer is that this is the fastest method, can be easily automated, and reasonably sensitive. Furthermore, this method does not destroy the protein. (3:56 - 4:46) However, many buffers and other reagents can interfere with the absorbance 280 spectrophotometric measurements. So the DNA and RNA have an absorbance maximum at 260 nanometer but still absorb at 280 nanometer and have tenfold higher absorbance values at 280 nanometer compared to the equivalent concentration of the protein. So therefore, if the sample is contaminated with nucleic acids, contaminated with DNA and RNA, the absorbance of 280 nanometer might not adequately reflect the real protein concentration. (4:47 - 7:17) Kasi nga maririd din yung mga nucleic acid. So the estimation of protein concentration is valid up to 20% nucleic acid or an absorbance at 280-260 nanometer ratio less than 0.6. So 0.6 po ang ratio lang dapat, less than 0.6. So the UV absorbance at 280 nanometer is also usable for the concentration determination if the sequence of the protein of interest contains a known amount of tryptophans and tyrosines. So the quantification of proteins by peptide bond absorption at 205 nanometer is more universally applicable than 280 nanometer. So 205 po. The absorptivity of a given protein at 205 nanometer is a several fold greater than the 280 nanometer. Thus, the lower concentration of protein can be quantified with the absorbance of 205 nanometer method. So 205 naman po ang gamit. So using this absorbance can be useful to quantitate the dilute solutions, short path length applications, continuous measurement ng mga column chromatography, tsaka yung mga aromatic amino acids. So this is the concentration limits for the reagents. Please be familiar po dyan ha, absorbance at 205 nanometer then absorbance at 280 nanometer. So another type under the UV spectrophotometry is using the intrinsic fluorescence emission. So usually the tryptophan fluorescence is measured as the indication of the fluorescence intensity of the protein sample solution. (7:17 - 8:06) So the concentration of the protein sample solution can be calculated by the calibration curve based on the fluorescence emissions. So normally the excitation wavelength is set to 280 nanometer and the emission wavelength is between 320 to 350 nanometer. Next is the colorimetric protein assay technique. So it detects the changes in color. So we detect the changes in color. So the ideal protein standard used in this quantitative assay is the exact same protein in a matrix or solution. (8:07 - 8:32) So yun po yung mga gamit natin. Ito yung mga gamit natin. Bavine serum albumin, gamma globulins, and immunoglobulins. So in practice there is not always a match protein standard available. So commercially available standards may be suitable for use is these three. Ito yung mga common na ginagamit. (8:35 - 8:51) So we have four methods for colorimetric protein assay. So we have the biuret reaction. So the purplish violet color, purple to violet color po ang output or product. (8:51 - 9:38) So when we observe the purple color, this is the formation of the cupric ions with proteins or yung mga peptide bands. So the biuret reaction with the proteins is independent of the composition of the protein. The next is the Lowry method. So we use phosphotangstic acid or phosphomolybdic as reagents. So this is Lowry plus phosphomolybdic or phosphotangstic acid. So known as the Follin-Shockal 2 reagent. (9:38 - 9:59) So ang product po ay blue-green color. So yung mga reagent na to class will interact with the copper or cuprous ions with the side chains of tyrosine, tryptophan, tsaka cysteine. So they produce a blue-green color po. (10:00 - 17:42) Next is the Bradford method. So this method is the easiest and fastest of the protein determination methods. Si Bradford method. So it uses a Kumasi Brilliant Blue G250 dye to proteins which results in a dye protein complex with increased molar absorption for the determination of protein. So yung product po nyan ay reddish or brownish color. So ang absorbance po na gamit ay 465 nanometer. So under acidic condition, ang dye ay magre-react sa mga arginine resulting to blue color in the wavelength ng 595. Then we have the bisynconinic acid assay. So based on the fact that the sodium salt of bisynconinic acid reacts with the cuprous ion generated by the bioret reaction under alkaline conditions. So the product is deep blue color at 562 nanometer wavelength. So yan po. Then we have the fluorescent dye base assay. So we have the microplate detection method. So this technique can be used to quantitate the proteins and peptides containing either the lysine or free nucleic acid terminal. So that we have three dyes used to quantitate proteins in this assay. We have the Opthalaldehyde, Fluoresamine, and 3,4-carboxybenzoylquinoline-2- carboxyaldehyde. So yan yung mga dye. Then next is the cuvette detection method. So in a cuvette based method class, the OPA reacts with the primary amino acid except cysteine in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent product. So yan po class. Yan yung column chromatography. So after the cells are broken and the cells extracts are released, proteins can be further purified through various biochemical methods. So they can be classified according to their size, their ion exchange, and affinity sa chromatography. So pag sa size class, size exclusion chromatography, they are separated based on their size and shape. So there's a column matrix, also known as resin, consists of microscopic beads of inert material. So class, in this type of column, small molecules, which is smaller than the cutoff threshold size, are more likely to go through the pores. Ito po yung mga pores ng matrix. Then they are trapped in the resin and travel through the column more slowly. Then on the other hand, yung mga larger molecules will move through the column then they are quickly, or they can move quickly because they cannot fit into the beads. So hindi sila matatrap dun class. Hindi sila makakapasok dun sa pores kasi malaki yung size nila. So the larger the molecules, so the large molecules will emerge first from the pores. So the larger the molecules, so the larger molecules will emerge first from the pores. So the larger the molecules, so the larger molecules will emerge first from the pores. Then yung small molecules will emerge last. Kasi sila nga yung matatrap sa resin. Kaya mas mauna yung mga larger molecules. Next is the ion exchange chromatography. So they separate the molecules based on the positive and negative charges. So if the beads in a column are positively charged, then the negatively charged proteins will bind to the column matrix at low salt as a result of ionic interactions. So the proteins can be induced to dissociate with high salt. Then in a low salt solution, proteins with a negative surface charge will bind more strongly to the positively charged and ion exchange columns. So likewise, proteins with a large, or with a positive surface charge will bind to negatively charged and ion exchange columns. So yung mga resins containing the positively charged groups attract to the negatively charged solutes. And these are referred as an ion exchange resin. So ito yung table po na yan. So resins with negatively charged groups are cation exchangers. Ito yung mga negatively charged groups. Then ito yung mga positively charged exchangers. Next is the affinity chromatography. So this is used to isolate the molecules based on the ligand binding abilities. So this is because in most of the instances, proteins carry out their biological activity through binding or complex formation with specific small molecules or ligands. So when the sample goes through the column, yung class yung column, elution column. So the protein of interest is displaying affinity for its ligand. So magkakaroon ng binding class. So the protein of interest is thus removed from the mixture of the sample. Then finally, the protein is dissociated or eluted from the resin by the addition of high concentrations of the free ligand in solution. (17:42 - 18:35) So class, matatandaan nyo class na yung parang ginawa natin sa laboratory. So yung unang pag nagmix yung protein sa ligands, then maglalagay tayo ng reagent, yung mga proteins magbabound or magbabind sa ligands. So ang malalabas po dun sa another tube or another tube or container is yung waste. Kasi nagbound na po yung protein sa ligand. Then another, mag-add ulit tayo ng reagent para naman bumaba yung target protein natin. So yun yung makakatch na dun sa another tube, sa new tube. (18:36 - 20:21) So the power of the affinity chromatography lies in the specificity binding of binding between the affinity reagent and the resin and the molecule to be purified. Molecular separation through the electrophoresis. So we have the SDS page or the sodium dodecyl sulfate polyacrylamide gel electrophoresis. So the hydrophobic tail of the SDS interacts strongly with the polypeptide chains. The number of the SDS molecules bound by a polypeptide or protein is proportional to the length of the polypeptide. So we have two advantages using this SDS page. So it coats all the polypeptides with negative charges and it masks the natural charges of the subunits. So after the reduction and denaturation by SDS, the proteins migrate in the gel according to the molecular mass. So class sa gel, transfer natin muna sa filter paper. Yan po yung magiging itsura nya. So the electrophoretic mobility of the proteins upon the SDS page is inversely proportional to the logarithm of the protein's molecular weight. So gel staining after the electrophoresis. So staining with the organic dye like comasi blue or comasi brilliant blue is the most frequently used. Transcribed by TurboScribe.ai. Go Unlimited to remove this message. 2024-11-27 22-38-59 Transcribed by TurboScribe.ai. Go Unlimited to remove this message. (0:03 - 1:37) The most frequently used for protein detection in SDS-PAGE method Okay, so the anionic trimethyl triphenylmethanedine is used to use in two modifications So we have Kumasi R-250 which is a red tint and Kumasi G-250 which is a green tint. So which has two additional methyl groups. In the presence of the acidic medium these dyes stick to the amino acids of the proteins by electrostatic and hydrophobic interaction. Next we have the zinc reverse staining. So it is based on the precipitation of the zinc imidazole in the gel zinc imidazole in the gel except in the zones where proteins are located. When zinc reverse staining is applied on a Kumasi blue stain gel previously undetected bands can be spotted. So what is not seen in the Kumasi blue staining can be seen using the zinc reverse staining So this technique is rapid, simple, cheap and reproducible and is and it is compatible with Microsoft analysis Next is the silver staining. We detect the proteins with silver because of the sensitivity below 1 nanogram per spot. And since the silver stain is cheaper, it is also used. (1:39 - 11:31) So silver staining is used as a second staining and essential to fix the proteins in the gel acidic alcohol prior to initial Kumasi blue staining So the fluorescence staining method is considered as endpoint method thus the results are highly reproducible so it require a fluorescence imaging or imager for visualization and it is a bit expensive compared to the classical colorimetric dyes. An example of fluorescence stain are Nile Red, Routinium Orange Red, Tangerine, Ruby Deep Purple, Cypro and also Epicochonone and Cyanide Then the gel-based proteinomics is the most popular and well-established technique for global protein separation and quantification. So through this technique class overall protein expression of tissues can be analyzed on a large scale and cheaper approach than gel-free proteomics So we have 2DE gel coupled with MS and Bioinformatics tools. So we have gel-based 2DE gel and 2DE fluorescence Differential imaging gel electrophoresis and gel-free proteomics like liquid chromatography and capillary electrophoresis So we have 2D or two-dimensional gel electrophoresis So the protein samples are separated into two dimensions according to their net charges at different pH and molecular weights determined by the SDS page Then the first dimension electrophoresis Okay We have conventional isoelectric focusing So the complex protein samples are separated according to their net charge at different pH So the mixture of proteins is electrophoresis through a narrow tube gel containing molecules called ampholytes So in your first step, the conventional then We immobilize the pH gradient So we have the immobilized pH gradient that is the integrated part of a polyacrylamide gel matrix fixed on a plastic strip. Then you have the non-equilibrium pH gel electrophoresis then the second dimension SDS page so Instead of loading the protein sample within the wells the First dimension is rehydrated strip is carefully placed on the top of the SDS page and sealed with agarose And we have modification of 2DE or two-dimensional difference gel electrophoresis differential imaging gel electrophoresis. It uses the size and charge match cyanine dyes With different excitation and emission wavelengths as protein labels for different samples So the sample buffer constituents for 2DE. We have the chaotrophic agents Detergents, reducing agents and carrier ampholytes. So please be familiar with the functions of those buffers Then we have gel-free proteomics techniques the capillary electrophoresis So this is a typical gel-free technique with the advantage of superior separation efficiency small sample consumption and short analysis time and Automatability So the protein separation in capillary electrophoresis is based on the properties of molecular mass, their isoelectric point and their electrophoretic mobility Then microchip electrophoresis. So the basic Capillary electrophoresis Configuration has been miniaturized and transferred to a chip format. So pinaliit siya Using the chip so with the Microchip electrophoresis, we can integrate the injection Separation and detection on a single microchip with typical channel lengths of 5 centimeter to 15 centimeter So remember here, microchip micro means small So smaller or lower voltages is what it needs and shorter the separation channels and Dahil small lang yung mga sample na kailangan Mas mabilis siya nagsaseparate or mas mabilis nagseparate yung mga proteins Okay, dahil micro lang yung mga sample So we have the liquid chromatography Useful in proteomics and genome research because it can detect the molecules at a nanomolar level So types of liquid chromatography, we have the reverse phase HPLC, affinity, gel permeation ligand exchange and capillary So we have the reverse phase HPLC. This is the most popular mode of chromatography Next is the affinity HPLC So There's a stationary phase Then dun nagbabind kasi mga molecules So nagtatrap dun klas, may stationary phase. Then we have gel permeation So the large molecules are eluted first class followed by the smaller molecules so based on their sizes gel permeation. Then we have the ligand exchange HPLC Either the anion or the cation exchange. So based on the ion exchange column, then we have the capillary electro chromatography so hybrid technique ng HPLC and capillary electrophoresis So After the proteins are separated and quantified the next step is to identify each protein in the sample So we have the mass spectrophotometer So this exploit the differences in the mass to charges ratio of ionized atoms or molecules So the basic operation of a mass spectrophotometer is to evaporate the ionized molecules in a vacuum creating a gas phase ions Separate the ions in space and or time base on their mass to charge ratio and measure the quantity of ions with specific mass to charge ratio Then we have the western blot. So another protein analysis. So this is a modification of southern blot class, etong western blot So And so the western blot probes are antibodies can be polyclonal or monoclonal Antibodies then bind to specifically to a immobilized target protein So the protein probes using western blot Applications may be labeled with 35s or the horseradish peroxidase or alkaline phosphatase Then after that we can confirm it using the ELISA For example class of this application The western blot method is confirmed of enzyme-linked Immunoassay ELISA for the HIV and hepatitis C virus So if there is a positive result class In ELISA, ELISA method for example in the test kit ELISA method is positive. For example in the HIV kit, ELISA is the Principle. When the result is positive, we confirm it using the western blot. So the western blot is made or made in San Lazaro So they are the confirmatory laboratory for sexually transmitted Infections and diseases Transcribed by TurboScribe.ai. Go Unlimited to remove this message.