Gel Electrophoresis and Blotting Techniques PDF

Lecture 1 Techniques for in vitro evaluation of biopharmaceuticals Gel Electrophoresis and Blotting Techniques Dr. Basma Nagy Abd El-Hamid  What is electrophoresis? - From the biological point of view: It is a technique that is widely used to separate purification nu...

Lecture 1 Techniques for in vitro evaluation of biopharmaceuticals Gel Electrophoresis and Blotting Techniques Dr. Basma Nagy Abd El-Hamid  What is electrophoresis? - From the biological point of view: It is a technique that is widely used to separate purification nucleic acids or proteins based on Charge, size and shape. - There are different types of electrophoresis, Zone electrophoresis.......including gel electrophoresis Moving boundary electrophoresis (e.g. capillary electrophoresis) Focus on gel electrophoresis  Principle of electrophoresis ++ + dna negative charge from phosphate groups repulsion rate loading of samples here occur separation here electric current pass immersed in buffer to conduct electricity. So When electric field was applied, negatively charged (DNA, Proteins) will migrate towards the anode (+ve charge).  Electric Field Battery Gel - Ve + Ve Gel are made of polymers such as Agarose or Polyacrylamide) immersed in buffer (to conduct electric field) If we placed the DNA, which is negatively charged (due to the phosphate group), on a gel , it will move towards the +ve end when the electric field is on.  Gel Electrophoresis Agarose gel Polyacrylamide gel electrophoresis electrophoresis (PAGE) (Vertical (horizontal electrophoresis) electrophoresis) Commonly used for Commonly used for separation of proteins separation of nucleic Can be formed of native acids PAGE and SDS-PAGE (Sodium neuclic acid has a larger size can migrate only from agarose gel dodecyl sulfate- PAGE) Both polymers form a matrix or mesh with certain pore size based on the type and the concentration of the gel  Generally Agarose is used for DNA separation?? imp As size of the DNA is lot bigger than proteins, a stable solid support with bigger pore size is required. Size of polyacrylamide pores is smaller for this purpose. Note: Agarose gel can also be used for separation of very large protein/protein complex. Agarose gel electrophoresis is most suitable for separation of DNAs/RNAs in the range of 100bp to about 15kb. Polyacrylamide gels matrix may be used for separation of small DNAs or RNAs. less than 15bp  Factors affecting migration The charge of substances plays a vital role in separation, While migration rate is mainly affected by other factors 1- Size or Molecular weight (protein) 2- Supporting medium (e.g. type of gel, Concentration of gel) 3- Conformation of nucleic acid.  1- Size The migration of the molecules in gel electrophoresis is inversely proportional to the size of the molecule. Small molecules migrate faster and then bigger ones as small molecules can move more easily through gel pores. Separation based on size The size of the fragments can be determined by running standard DNA/protein ladder run in parallel. ladder dna segments 200  2- Gel concentration Migration rate of the DNA fragments also depends on the concentration of agarose used to prepare gel. The concentration of agarose is inversely proportional to the rate of migration of the DNA fragments. higher conc decrease size of pores and so seperated effeciently. Lower the concentration.....the faster is the DNA migration rate and vice versa.  3- Conformation or shape The linear relationship between size and migration is applicable only for linear DNA fragments. But DNA can exist in three forms: linear form, opencircular form and supercoiled form. Therefore, for the same size plasmid DNA, supercoiled DNA form > faster than open circular form > linear form.  1- Agarose gel electrophoresis Agarose gel matrix Which is composed of agarose sugar. Which can crosslink together through hydrogen bonds form porous matrix, its pore size based on concentration of agarose solution  How to perform Agarose gel electrophoresis A- Preparation of gel (Gel casting) Components: 1- Agarose 2- Tris/borate / EDTA buffer, TBE (pH8) or Tris/acetate/EDTA buffer, TAE) 3- Visualization dye (Ethidium bromide (EtBr)/ cyber green) Highly carcinogenic, dangerous 99999 Ethidium bromide is a florescent dye that intercalates between bases of nucleic acids and allows convenient detection of DNA fragments in agarose under UV light. To Dissolve agarose in TBE buffer 2- Boil in microwave, 3- Once dissolved and cool down to 60 °C , add visualizing dye (0.5 ug/ml EtBr. 4- Pour it in gel cassette. 5- A comb can be inserted into the hot agarose to cast the well for loading the sample sample and dna ladder 6- Gel allowed to cool at RT, for about 1 h to solidify the gel, then comb was removed gently 7- The gel is placed in the buffer tank (same TBE buffer) carefully with buffer completely submerge the gel ----  B- Running gel Load your DNA samples in corresponding wells in the gel B1-How to prepare sample? DNA sample (extracted from the cell) +Loading buffer +Restriction enzymes (endonucleases) Restriction enzymes cut DNA in very specific place , each one detect specific sequence of DNA and cut down stream the sequence.  So we should use endonucleases specific to the sequence of my interest  We have some DNA that has not been cut,  some been cut by the restriction enzyme (gene of interest), other fragments.  In one sample we have what is called a ladder. In this sample are fragments of known size so we can use it as a kind of ruler to get an idea of what size our fragments in our other samples are.  B2-Loading buffer It is composed of : Glycerol: used to increase the viscosity of the DNA sample to settle down in the well other wise will mix with the buffer and lost. Coloring dye (e.g.bromophenol blue): used to dna give a color to the sample so we detect sample while migrate and can turn power off after sample reach end of the gel. then why add dye to the gel again ?  B- Running gel Sample Loaded Power turn on , DNA fragments migrate through the gel  C- Visualizing the DNA fragments When a gel is stained with a DNA-binding dye (e.g. EtBr) and placed under UV light, the DNA fragments will glow, allowing us to see the close to 500 DNA present at different so it may be 600 locations along the length of the gel. 1. Ethidium bromide: Fluorescence at certain wave length and can bind to DNA and RNA 2. Cyber Green: bind only with DNA (double stranded only).  2- SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Vertical electrophoresis It is used for protein separation. Polyacryamide gel (inert material)  SDS-PAGE gel discontinuous gel two types of gel collect protein molecules - p --- 2- Stacking gel (pH 6.8) Battery 1- Resolving gel or separation + gel (pH 8.8) A- Casting SDS-PAGE gel “Equipments used” Casting frame Long glass plate Short glass plate Casting stand  Gel components Both resolving gel and stacking gel consist of :  Acrylamide/bisacrylamide (solution A)  Tris-HCl/SDS buffer, (1.5 M pH 8.8 for resolving gel (solution B), 0.5 M pH 6.8 for stacking gel (solution C)  Ammonium persulphate (APS): it is an initiator for the acrylamide polymerization  TEMED: It forms oxygen free radicals, induce polymerization of the acrylamide , once added to gel must be casted immediately, so we add it at the end  Acrylamide Amm. persulfate TEMED Free radical initiator Accelerates formation of free radical from persulfate Induce Polymerization Bisacrylamide Cross-Linking pores size depend on conc and make cross link between ratio of acrylamide and polymers so formation of bisacrylamide mesh or pores so each specific size need specific conc of them  Different One conc. of For example stacking gel concentration of separating gel Separating Stacking acrylamide conc x% 18% 15% 12% 9% 7% 5% A x/3 ml 6 5 ml 4 ml 3 ml 2.33 ml 0.67 ml B 2.5 ml 2.5 2.5 ml 2.5 ml 2.5 ml 2.5 ml - C - - - - - 1.0 ml H2O 7.5-x/3 1.5 2.5 ml 3.5 ml 4.5 ml 4.67 ml 2.3 ml 10 APS 50 ul 50 ul 50 ul 50 ul 50 ul 50 ul 30 ul TEMED 5 - 10 ul 5ul 5 ul 5 ul 5 ul 10 ul 5 ul Total 10 ml 10 ml 10 ml 10 ml 10 ml 10 ml 4 ml  --- A- Gel casting 2- Add 1- Add layer of resolving gel isopropanol between 2 to remove plates Resolving gel bubbles ---- 4- Add comb to perform wells Wait until dry directly after adding of stacking gel then remove isopropanol using filter paper 5- Wait until dry 3- Add use the prepared gel same Stacking gel, day or save wrapped over load to inside cassette in 4°C till remove air needed bubbles  Isopropanol layer to stop the entry of oxygen (oxygen neutralizes the free radical and slow down the polymerization) and make the top layer smooth  B- Sample preparation Denaturation buffer or mix or purified Protein sample buffer or Lamellae buffer Denaturation buffer: composed of Tris-Hcl (pH 6.8), SDS, β- Mercaptoethanol, glycerol, bromophenol (blue dye)  Hydrogen bonding Disulfide Protein O bonding Ionic bonding Hydrophobic bonding SDS Mercaptoethanol - - - - - - Separation based on M. wt. or size shape not affect separation  SDS β-Mercaptoethanol Anionic surfactant Its function: It help also in  Bind to amino acids side denaturation of protein chains giving the It is a reducing agent, protein net negative can reduces disulfide charge bonds in the proteins  Break non covalent bond in proteins cause protein unfolding *So the shape of protein does not play a role in SDS- PAGE as well as the charge. *Only molecular weight of proteins responsible for separation  C- Running of SDS-PAGE gel Running buffer (electrophoresis buffer), pH 8.3 Composed of Tris/glycine/SDS buffer agrose we used one type of buffer and gel but here use 2 gel (resolving, stacking) 3 buffer (buffer 8.8, buffer 6.8 (sample, stacking), running 8.3 with different component glycine)  − Stacking gel, pH 6.3 Running buffer Ensure all pH 8.3 proteins arrive Separating gel separating gel at pH 8.8 the same time +  − − − − − − − − − One Running buffer − −− − Well pH 8.3 − No Stacking gel Separating ----- gel pH 8.8 + The pH of the stacking gel is 6.8 and at this pH, glycine is moving slowly in the front where as Tris-HCl is moving fast. As a result, the sample gets sandwiched between glycine-Tris and get stacked in the form of thin band below Above pH 6 pH 6 PKa1= 5.95 PI= 5.95 PKa2= 9.6  − Glycine − One Glycine − Running buffer Glycine − Well pH 8.3 Cl − Cl − Cl − Cl − − − − − − − − − − − − − − here the stacking ph 6.8 glycine contain low negative Stacking gel charge so migrate slower than the proteins forming sandwish pH 6.8 pH ≈ PI +  − Glycine − Glycine − Glycine − − − − − − − − − − − − − − Cl − Cl − Cl − Cl − in separating ph 8.8 glycine highly negative charge so Separating gel emigrate rapidly and then protein pH 8.8 molecules by size we used this stacking gel in this method as it is vertical and pH > PI gravity can affect the migration +  D- staining the gel Remove gel carefully by sliding with H2O and rinse to remove SDS. Add 40 % EtOH: 10% Acetic acid......shake for 15 min.....fix the gel. Add coomassie blue stain( directly on gel).....overnight specific for protein only Destain, to get rid of the background (rinse with water repeatedly for 3h).  E- Gel analysis “Bioimaging system UVP”  Image of the gel by program (e.g. azure Biosystems.  Analyzed using software (e.g. image J or Azure spot analyzing software (bio imaging system UVP) Ladder can be used for identifying Molecular weight of protein. Standard protein known concentrations can be run with unknown to quantify the unknown through calibration curve  Agarose versus SDS-PAGE pore size based on the conc gel in both types Agarose SDS-PAGE Gel made of long chains of agarose PA is made by chemical interlinked sugars to create crosslinking of acrylamide and meshwork. bis-acrylamide. This linking produces a sieve. Gel casted horizontally Gel casted vertically Continuous gel (running gel) Discontinuous (stacking , running gel) Non toxic potent neuro-toxic Separate large molecules, Separate small molecules, commonly commonly used for DNA used for Protein separation. separation Staining for visualization can be Staining for visualization can be done done before pouring the gel After pouring the gel  Blotting Techniques Blotting techniques are used to identify unique proteins or nucleic acid sequences. highly specific and sensitive and have become important tools in both molecular biology. a- Western b- Southern c- Northern Blotting Blotting Blotting Specific Protein Specific DNA Specific RNA  General Principle probe substace genes of interest forming solid lines and cancel other genes 1- Electrophoretic separation of protein or of nucleic acid fragments in the sample 2- Transfer to and immobilization on paper support. 3- Binding of analytical probe to the target molecule on paper 4- Visualization of bound probe.  Western Blotting (Immunoblotting) most complicated use antibody Technique to detect specific proteins in protein mixture. e.g. Protein extract from cell........Protein mixture..... interested to detect specific protein in this mixture. Step 1: Electrophoresis (In this case, SDS-PAGE)  Why we don’t analyze the gel directly as previously mentioned??? I. In each sample......may have hundred of proteins because it is extracted for example from a cell. Just one protein of these is my interest. So it is impossible to see this protein in the gel II. So in case of blotting...... I have specific antibody that have specific binding site to the protein of my interest. Antibody can not bind to the protein inside Protein the gel inside the gel Specific Antibody  So we have to transfer the protein to a surface (blotting) where the antibody can detect and bind to it  Steps of western blotting A- Transfer of the protein to a membrane  Nitrocellulose membrane (Commonly used membrane) or other nylon membranes  Has affinity to protein so proteins can bind to its surface.  Polyvinylidene fluoride (PVDF) membrane. Nitrocellulose Gel obtained Western membrane after Blotting electrophoresis Sponges Sponges: to compress on the gel and membrane Filter paper Filter paper  Western Blotting + − Transfer buffer: Tris base (pH 8.3) Glycine Methanol glycine will be highly -ve charge so filter paper (capillary action) to will move first then proteins make the gel and membrane wetted by buffer soln only Apply electric current (battery), -ve on gel side and +ve to membrane side  A- Transfer to membrane Methanol in the buffer: can detach SDS molecules from protein to facilitate protein to transfer to membrane  B- Immuno-detection cellulose membrane has affinity to bind to the antibodies , so it is blocked by free fat milk cover all the membrane except the target area The Western blot is blocked, incubated with antibodies, and treated with substrate to make the target protein visible. Wash steps are carried out between incubations to remove excess unbound material and to minimize non- specific signal on the immunoblot  B- Immuno-detection After blotting, the target protein will be detected using appropriately matched and labeled antibodies. The typical immunodetection stage involves a few basic steps: 1- Blocking - The blot containing the transferred protein bands is incubated with a protein or detergent solution which covers the entire surface so that antibodies do not bind non-specifically to the membrane. (e.g. we can use fat free milk)  2- Antibody incubation - Labeled antibody binds to the target protein band present on the blot in a one-step or two-step procedure. (1ry Ab)(Ab can be commercialized or extracted, not labelled).....then apply 2ry Ab (labelled to detect the 1ry Ab). to magnify the result or detection when the sample is small advant that two or more labled antibodies bound to one antibody so magnify the detection. 3- Detection with substrate (Chemiluminescence) - The label attached to the antibody, usually an enzyme is detected using a substrate which produces a visible signal corresponding to the position of the target protein.  Southern and Northern blotting General Steps Need to detect specific sequence of DNA or RNA Gene of my interest.... Should know sequence of this gene......design complementary sequence (probe) This probe can detect gene of my interest. So to do this we have to transfer the genes to membrane to be free to bind to probe. This can not be done on the gel  A- Transfer buffer contiune to paper and In the paper will southern and +ve absorb the exss Northern blot, charge , high Transfer done by affinity to dna capillary transfer conduct (against the gravity) buffer to gel to deliver dna not electrical transfer seq to membrane (as in western blot) DNA (-ve) will stick on the nylon (+ve) membrane through ionic exchange forces Nitrocellulose membrane with adsorbed DNA fragments.... Bake at 80° C for 2h or UV radiation to Fix DNA in NaOH soln before transfer to membrane attach probe to dna seq  Probes a) Definition: is a single strand of DNA that can hybridize (base pair) with a complementary sequence on another single stranded DNA or RNA. b) The probe must contain a label so that it can detect complementary DNA or RNA. The label may be radio active material (e.g. radioactive phosphorus, 3 3P), so it can be detected by autoradiography or chemical that can be identified, for example by fluorescence  Hybridization Special radioactive labeled probes having complementary bases to the specific gene searched for, are then applied to the membrane where they will combine with the gene.......Gene detection.  Autoradiography It is the detection of radioactive molecules (e.g. DNA, RNA and protein) by visualization of their effects on photographic films Or the probe can be fluorescently labeled and detect under dark chamber.  Northern blotting?? Same as Southern blotting (DNA) but for RNA Search for RNA sequence??? to see if gene (I know is present) is expressed or not (over or not expressed) RNA always present in 2ry structure.....so denature 2ry structure to obtain linear RNA which can bind to the probe. This can be done by adding Formaldehyde to agarose gel during electrophoresis  for denaturing Membrane pretreatment 2ry antibody