GENETIC TEACHER

To Assemble Polyacrylamide Gel Apparatus: 1- Prepare polyacrylamide gel, 2- Add diluted samples to the sample buffer, 3- Heat them to 95 degree Celsius for 4 minutes, 4- Load the samples onto polyacrylamide gel, 5- Run the gel apparatus at 200 volts for 30 to 40 minutes, 6- Stain the gel with standard stain #geneticteacher

SDS-PAGE Casting Materials: 1- casting stand, 2- casting frame, 3- casting stand gasket, 4- comb, 5- gel releaser #geneticteacher

Peptide markers range from 2.5 to 17 kilo Dalton, low molecular weight range from 14 to 94 kilo Dalton, high molecular weight rang from 53 to 212 kilo Dalton, rainbow markers rang from 10 to 250 kilo Dalton #geneticteacher

Dye for tracking progress of electrophoresis are: Bromophenol Blue (Fasting migration) and Xylene Cyanole (Slow migration) #geneticteacher

Dense material, carry sample to bottom of gel such as (Sucrose, Glycerol or Ficoll) #geneticteacher

TBE or Tris-Borate EDTA, is high buffering capacity with pretty good resolution #geneticteacher

TAE or Tris-Acetate EDTA, is a good resolution of DNA but buffering capacity is quickly depleted #geneticteacher

Stocks of gel solutions of SDS-PAGE: Stock 1 (30 gm acrylamide + 0.8 gm Bis-acrylamide) complete total volume to 100 ml distilled water, Stock2 (18.2 gm Tris-base or Tris-HCL dissolved in 50 ml distilled water) complete total volume to 100 ml distilled water with pH= 8.8, Stock 3 (6 gm Tris-HCL dissolve in 50 ml distilled water) complete total volume to 100 ml distilled water with Ph= 6.8 #geneticteacher

Electrophoresis proteins in the gels stain by Coomassie blue dye for 1 hours with agitate slowly on shaker, and destain the gel in destaining solution few times until protein bands visualized, then the gel photographed and transferred to preserving solution containing 10% glycerol in destaining solution for an hour and allow to dry at room temperature when the surface of the gels appeared sticky, or mount the gel between cellophane preserving sheets and left overnight for through drying at room temperature #geneticteacher

In SDS-PAGE protocol: 15 % acrylamide concentration separate (10 – 43) KDa, 12 % acrylamide concentration separate (12 – 60) Kda, 10 % acrylamide concentration separate (20 – 80) Kda, 7.5 % acrylamide concentration separate (36 – 94) Kda, 5 % acrylamide concentration separate (57 – 212) KDa #geneticteacher

The top of the gel box in SDS-PAGE attach to negative power outlet, and the bottom of the gel box attach to positive power outlet, so movement through PAGE gel is proportional to mass not to charge #geneticteacher

SDS denature protein and covers it with negative charges moves to positive end #geneticteacher

Due to high density of binding SDS to proteins, the ration (Size/Charge) is nearly the same for many SDS denatured proteins, hence proteins are separated only by length of their polypeptide chain not by difference in charge, so great separation allows estimation of size of polypeptide #geneticteacher

SDS-PAGE, is sodium dodecyl sulfate polyacrylamide gel electrophoresis, separates proteins based primarily on their molecular weight in electric filed by uses a discontinuous gel as a support medium and SDS to denature proteins #geneticteacher

SDS-PAGE gel, form between two glass plate, the larger plate has precisely milled spacer at either edge that generate 1 mm separation between assembled plates, while the second plate is shorter and has the same width as a spacer plate, and make sure plate are clean before proceeding with taking care with its fragile #geneticteacher

Protein extracted by grinding 1 gm freeze dried sample in pestle and mortar with liquid nitrogen to crush samples, the electrode buffer is placed on them and transferred to Eppendorf tube, boiled for 10 minutes and centrifuge at 10000 rpm to take the supernatant as isolated protein,, then estimate the protein content in supernatant with bovine serum albumin as standard protein, then adjust protein content to 2 mg /ml #geneticteacher

Separating gel, pour between glass sandwich using scientific instruments and gently covered with 1 cm of water, and polymerization start within (25 – 30) minutes, then the stacking gel pour and allow for polymerization in about 30 minutes, after that add running buffer to upper and lower reservoirs #geneticteacher

There are two types of PAGE: 1- Native-PAGE, separation based upon charge, size and shape of macromolecules, so it useful for separation and purification of mixture proteins, 2-Denature-PAGE or SDS-PAGE, separation based upon molecular weight of proteins, so it consider the most common method for determining molecular weight of proteins, therefore, it consider very useful for checking purity of protein samples #geneticteacher

Native proteins may be put into gel systems and electrophoresis because alternative native proteins gel forces all proteins to acquire the same size to charge ratio #geneticteacher

Proteins unlike DNA don’t have constant size to charge ratio, so, in an electrical field, some move to positive pole and some move to negative pole, and some not move because they are neutral protein #geneticteacher

In SDS-PAGE protocol, Turn of power before the tracking dye runs off the gel #geneticteacher

To load sample in SDS-PAGE protocol: Place a loaded gel loading tip at the bottom of sample well and slowly expel the sample by avoiding air bubbles #geneticteacher

Destaining buffer for SDS-PAGE protocol: 100 ml acetic acid glacial + 400 ml methanol + 400 ml distilled water #geneticteacher

Staining buffer for SDS-PAGE protocol: 1 gm Coomassie brilliant blue R + 180 ml methanol + 180 ml distilled water + 40 ml acetic acid glacial #geneticteacher

Loading Dye of SDS-PAGE: (0.005 gm bromophenol blue + 12 ml glycerol) dissolved in 8 ml distilled water for total volume 20 ml #geneticteacher

Function of Running or separating gel in SDS-PAGE, is the actual size of separation protein samples based on their mobility , because separating or running gel has pH =8.8 to separates proteins on the basis of size #geneticteacher

Function of Stacking gel in SDS-PAGE, is concentrate the proteins sample in sharp band by ordering and arranging protein bands before entering the field of separation gel, because stacking gel has pH= 6.8 with low polyacrylamide concentration, focuses protein into thin layer to give higher resolution upon separation #geneticteacher

Electrophoresis, migration of charged particle through separation matrix toward positive charged electrode #geneticteacher

Coomassie blue stain, detect (10 – 50) ng protein per band, Silver stain, is 50 times more sensitive than Coomassie blue and detect (0.3 – 0.5) ng protein per band, Fluorescent stains, almost sensitive than silver stain and required excitation sources #geneticteacher

SDS-PAGE destained by immersion the SDS-PAGE gel in a destaining solution until the background be clear, the destain solution is (25% methanol + 10 acetic acid + distilled water) at room temperature #geneticteacher

Coomassie Blue protein binding, interacts with positively charged R groups in its anionc form by fix the gel in a filtered Coomassie blue stain overnight containing (0.1% Coomassie brilliant blue R dissolved in 30% methanol + 10% acetic acid + water) at room temperature #geneticteacher

Discontinuous SDS-PAGE, has stacking gel that is layered on top of separating gel (High Resolution) #geneticteacher

Continuous SDS-PAGE has only single separation gel and uses the same buffer in the tanks of SDS-PAGE (Less Resolution) #geneticteacher

To Make Fresh SDS 10% for SDS-PAGE: 0.5 gm SDS dissolved in 5 ml distilled water #geneticteacher

TEMED in SDS-PAGE, use to accelerate free radical generation by Ammonium Per Sulphate #geneticteacher

To Make Fresh Ammonium Per Sulphate (APS) 10% for SDS-PAGE: 0.5 gm APS dissolved in 5 ml distilled water to generate free radicals needed for polymerization #geneticteacher

Electrode buffer of SDS-PAGE, (6 gm Tris Base + 28.84 gm Glycine + 2 gm SDS) and complete volume to 1000 ml distilled water to make 2X solution #geneticteacher

Samples buffers of SDS-PAGE: 7.88 gm Tris-HCL dissolved in 50 ml distilled water to make solution 1 molar Tris-HCL with pH= 6.8, then, mix them with (1 gm SDS + 2.5 ml Tris-HCL 1 molar) dissolved in 40 ml distilled water and complete total volume to 50 ml #geneticteacher

Contents of samples buffer of SDS-PAGE: 1- SDS, denature proteins and makes them negatively charged, 2- B-mercaptoethanol, use to break disulphide bonds, 3- Glycerol, increase density of sample relative to surrounding running buffer making it easier to load in well, 4- Bromophenol blue, use to follow the run proteins samples on gel as a tracking dye #geneticteacher

Components of SDS-PAGE gel contains, 1- Stacking gel, large pore polyacrylamide, 2- Resolving gel, small pore polyacrylamide gel resolves protein (24 – 205) KDa, 3- Running buffer, run through gel slower than slowest protein at pH above 8 #geneticteacher

Applications of SDS-PAGE: 1- measuring molecular weight, 2- Peptide mapping, 3- Estimation of protein size, 4- Determination of protein subunits of aggregation structure, 5- Estimation of protein purity, 6- Protein quantification, 7- Monitoring protein integrity, 8- Comparing polypeptide composition of different samples, 9- Analysis of number and size of polypeptide subunit, 10- Post electrophoresis application such as western blot #geneticteacher

Acrylamide, is monomers and toxic polymerized to form gel matrix for separation nucleic acids (6 – 1000) bp and useful in separation proteins when proteins are treated with SDS to gives uniformly negative surface charge #geneticteacher

PAGE, is a backbone of blotting techniques and very high resolution than agarose #geneticteacher

Polyacrylamide, forms much more restrictive gel and is mechanically and chemically more stable and clearly than agarose #geneticteacher

Agarose gel, use for protein electrophoresis in clinical diagnostic for analysis of large proteins over 800 kDa #geneticteacher

Results of multiple sequence alignment of clustal W appear as a result files including(Input sequences, Tool Output, Alignments in Clustal Format, guide tree) and scores table #geneticteacher

Custal W tool is available at (EMBL-EBI web site) as a online tool of sequence analysis including sequence input needed #geneticteacher

Clustal W software has three steps process: 1- construct pairwise alignments, 2- build guide tree by neighbor joining method, 3- progressive alignment guided by tree by aligned sequences progressively according to branching order in the guide tree #geneticteacher

Clustal W software, is a most popular multiple alignment tool stands for weighted which mean different parts of alignments are weighted differentially #geneticteacher

Clustal X is a statistical software use to arrange molecules Data (DNA or RNA or Proteins) to search for point mutations to facilitate future development of the alignment algorithms, after download clustal X software, open it, and select file menu for load sequence file, Sequence file that be used for loading in clustal X must be excel file, there are several point mutations between aligned sequences ., these mutations are (deletion., insertion., and substitution)