Human Cell Biology Lecture 2 PDF

BIOL 4130H Human Cell Biology Lecture 2 Cellular Approaches and Techniques 2 Lecture Outline 1. Antibodies revisited 2. SDS-PAGE and western blotting 3. Expression vectors 4. Centrifugation Antibodies Revisited ▪ We generate antibodies by injecting an antigen into...

BIOL 4130H Human Cell Biology Lecture 2 Cellular Approaches and Techniques 2 Lecture Outline 1. Antibodies revisited 2. SDS-PAGE and western blotting 3. Expression vectors 4. Centrifugation Antibodies Revisited ▪ We generate antibodies by injecting an antigen into a host animal ▪ The antigen can be a small synthetic peptide (10-20 aa) based on the protein sequence, a larger fragment of the protein sequence, or the entire protein sequence ▪ Polyclonal and monoclonal antibodies detect epitopes within the antigen that are 4-6 aa in length ▪ Polyclonal antibodies contain a mixture of antibodies that detect different epitopes within the antigen ▪ Monoclonal antibodies detect a single epitope within the antigen Antibodies Revisited ▪ Benefits of using a polyclonal antibody (compared to a monoclonal antibody): ▪ Inexpensive and relatively quick to produce ▪ Higher affinity to antigen ▪ Easier to detect the native protein ▪ Drawbacks: ▪ Limited amount ▪ Host-to-host variability ▪ Greater chance of cross-reactivity with other proteins Antibodies Revisited >DDB0233997|DDB_G0283921 |Protein|gene: ctsB on chromosome: 4 position 1297288 to 1298315 MRVLLSLVVILFIINSAFAVKINIGRPTKSHKTIHHETWVEEQTDQFDNIKVGQLLGFKR SPNRPKLQIKSYDPLGVQIPTSFNAQTNWPNCTTISQIQNQARCGSCWAFGATESATDRL CIHNNENVQLSFMDMVTCDETDNGCEGGDAFSAWNWLRKQGAVSEECLPYTIPTCPPAQQ PCLNFVNTPSCTKECQSNSSLIYSQDKHKMAKIYSFDSDEAIMQEIVTNGPVEACFTVFE DFLAYKSGVYVHTTGKDLGGHCVKLVGFGTLNGVDYYAANNQWTTSWGDNGTFLIKRGDC GISDDVVAGLP* >DDB0233997|DDB_G0283921 |Protein|gene: ctsB on chromosome: 4 position 1297288 to 1298315 MRVLLSLVVILFIINSAFAVKINIGRPTKSHKTIHHETWVEEQTDQFDNIKVGQLLGFKR SPNRPKLQIKSYDPLGVQIPTSFNAQTNWPNCTTISQIQNQARCGSCWAFGATESATDRL CIHNNENVQLSFMDMVTCDETDNGCEGGDAFSAWNWLRKQGAVSEECLPYTIPTCPPAQQ PCLNFVNTPSCTKECQSNSSLIYSQDKHKMAKIYSFDSDEAIMQEIVTNGPVEACFTVFE DFLAYKSGVYVHTTGKDLGGHCVKLVGFGTLNGVDYYAANNQWTTSWGDNGTFLIKRGDC GISDDVVAGLP*  Immunofluorescence – Detecting Multiple Proteins in the Same Cell ▪ Rabbit polyclonal anti-Cdk detected with goat polyclonal anti-rabbit Alexa 488 ▪ Mouse monoclonal anti-tubulin detected with donkey polyclonal anti-mouse Alexa 555 Huber and O’Day, 2011 Methods in Cell Biology ▪ Microscopy—use light and electron microscopy to image cells/organisms ▪ Electrophoresis—use an electrical field to move DNA, RNA, or proteins through a medium (e.g., agarose, acrylamide) based on size and charge ▪ Subcellular fractionation—use centrifugation to separate/isolate different organelles and macromolecules ▪ Mass spectrometry—use mass spectrometers to determine the composition of a sample  SDS-PAGE ▪ Proteins can be separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) ▪ SDS-PAGE minimizes differences in charge among the separated proteins © 2016 Pearson Education, Inc.  SDS-PAGE © 2016 Pearson Education, Inc. SDS-PAGE ▪ It is also common to add a reducing agent such as dithiothreitol (DTT) or β-mercaptoethanol (BME), which reduces disulphide linkages ▪ Since SDS coats proteins with a uniform negative charge, the total charge on the denatured proteins is directly related to their length ▪ Thus, proteins are separated based on their size ▪ Larger proteins = ↓ concentration of polyacrylamide ▪ Smaller proteins = ↑ concentration of polyacryamide  SDS-PAGE ▪ To determine the sizes of proteins in a sample, one lane in the gel is loaded with molecular weight (MW) standard (or ladder), which contains an assortment of purified proteins of known size (expressed in kDa) ▪ Proteins in the gel can be stained © 2016 Pearson Education, Inc.  SDS-PAGE Silver-stained gel Gel stained with Coomassie Brilliant Blue dye © 2016 Pearson Education, Inc. Huber and O’Day, 2015 Western Blotting ▪ SDS-PAGE will only provide information about the molecular weights of the separated proteins ▪ To study a specific protein, we need to perform a western blot ▪ Q: Why is it called western blotting?  Western blotting © 2016 Pearson Education, Inc.  Revealing Proteins on Membranes ▪ Before the transfer, you can also stain your membrane with Ponceau S stain to reveal proteins ▪ Ponceau S staining is reversible and can be removed with a short incubation in 0.1% NaOH ▪ Used to assess equal loading https://www.thermofisher.com/order/catalog/product/A40000279 Steps in Western Blotting 1. Incubate the membrane in 5% skim milk or 5% bovine serum albumin (BSA) to block non-specific sites on the membrane 2. Incubate the membrane (time varies) with a primary antibody against the protein of interest 3. Wash the membrane for 30 mins Steps in Western Blotting 4. Incubate the membrane (time varies) with a secondary antibody directed against the primary antibody Secondary antibody linked to horseradish peroxidase (HRP) Secondary antibody must be designed in a different host from the primary antibody (e.g., mouse monoclonal anti-rabbit HRP, rabbit polyclonal anti-mouse HRP) Steps in Western Blotting 5. Wash the membrane for 30 min 6. Incubate the membrane with enhanced chemiluminescence (ECL) reagent HRP acts on the substrate in the chemiluminescence reagent (luminol) to emit light at 428 nm 7. Expose the membrane to film or use an imager that can detect the chemiluminescence  Steps in Western Blotting https://www.antibodies.com/applications/western-blotting Steps in Western Blotting ▪ Film exposure ▪ Not used much anymore ▪ Film is expensive ▪ Not as sensitive as other methods ▪ Difficult to determine the ideal exposure time Huber, unpublished Steps in Western Blotting Steps in Western Blotting Huber et al., 2020 Steps in Western Blotting ▪ Western blotting requires loading controls (e.g., actin, tubulin, GAPDH) ▪ Q: Why do think this is necessary? Huber et al., 2017 Working with DNA ▪ Recombinant DNA technology: Uses restriction enzymes to cut DNA at specific places allowing scientists to create recombinant DNA molecules with DNA from different sources ▪ DNA cloning: Generates many copies of a specific DNA sequence ▪ DNA transformation: Introduces DNA into cells ▪ DNA sequencing: Determines the nucleotide sequences of DNA Expression Vectors ▪ What if an antibody against your protein of interest is not available? Can you still study its subcellular localization? ▪ The answer is YES! ▪ Using expression vectors, we can tag a protein with a fluorescent (e.g., GFP) or non-fluorescent tag (e.g., FLAG, HA, His, myc) ▪ Expressed proteins can also be purified and studied biochemically (e.g., enzyme assays, immunoprecipitation, etc.) Expression Vectors – Fluorescent Tag ▪ DNA encoding a protein of interest is fused with DNA encoding a portion of another protein that fluoresces ▪ When introduced into the cells of an organism, the protein produced from this DNA is visible within living cells ▪ Green fluorescent protein (GFP) from Aequorea victoria is commonly used ▪ Variants of GFP that fluoresce blue (BFP), yellow (YFP), and cyan (CFP) are also available that were generated through directed mutagenesis of the GFP gene N-term vs. C-term GFP Steps in Generating a Construct to Express a Fusion Protein 1. Amplify your gene of interest from cDNA Why cDNA? 2. Digest the ends of gene X (i.e., 5’ and 3’) and the expression construct with appropriate restriction enzymes 3. Ligate the two pieces of DNA 4. Transform this ligation product into competent bacteria  Steps in Generating a Construct to Express a Fusion Protein https://www.neb.com/en/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future Steps in Generating a Construct to Express a Fusion Protein 5. Screen the bacterial colonies to determine which colony carries the desired construct (i.e., the construct that contains gene X) 6. Isolate the expression construct from the bacteria using commercially available kits (i.e., mini-prep) 7. Transform, transfect, or transduce your cell line of interest with this expression construct  Live Cell Imaging of Dictyostelium Cells Expressing GFP-Cln3 Huber et al., 2014 N-term vs. C-term GFP N-term vs. C-term GFP ▪ The GFP tag can be fused to either the N-term or C-term of a protein ▪ Why do you think it might be important when studying an uncharacterized protein to study fusion proteins that contain GFP on either the N- term or C-term of the protein? ▪ E.g., GFP-geneX and geneX-GFP  N-term vs. C-term GFP Huber and Mathavarajah, 2018  N-term vs. C-term GFP Huber and Mathavarajah, 2018 Expression Vectors – Non-Fluorescent Tag 1. Generate a construct containing Gene X and the fusion tag of interest (e.g., FLAG) 2. Transform, transduce, or transfect this construct into the desired cell line 3. Fix the cells (e.g., methanol, paraformaldehyde) Expression Vectors – Non-Fluorescent Tag 4. Primary antibody against the fusion tag (e.g., anti- FLAG) 5. Secondary antibody linked to a molecule that fluoresces (e.g., FITC, Rhodamine, Alexa 488, Alexa 555, etc.) 6. Image the cells using fluorescence microscopy  Expression Vectors – Non-Fluorescent Tag HeLa cells non-transfected (A) or transfected with a FLAG- fusion protein (B and C) were fixed with 4% PFA and stained with Alexa Fluor® 488 anti-DYKDDDDK antibody. Cells were also stained with DAPI to reveal nuclei (blue). https://www.biolegend.com/en-us/search-results/alexa-fluor-488-anti-dykddddk-tag-15653  Using Centrifugation to Isolate Organelles ▪ Each organelle has a characteristic density based on its unique composition ▪ When a solution is spun in a centrifuge, the particles within the solution will migrate through the solution based on size and density as well as the solution’s density and viscosity © 2016 Pearson Education, Inc. Using Centrifugation to Isolate Organelles ▪ Subcellular fractionation allows researchers to isolate and purify specific organelles and macromolecules ▪ Step 1: Homogenize the tissue ▪ To preserve the integrity of organelles, homogenization is usually done in a cold isotonic solution such as 0.25M sucrose. Q: Why? ▪ Tissue can be homogenized using a variety of approaches (e.g., mortar and pestle, enzymatic digestion, cell lysis, etc.). Using Centrifugation to Isolate Organelles ▪ Step 2: Differential centrifugation ▪ This procedure separates organelles and other cellular components based on their differences in size and density ▪ The relative size and density of an organelle or macromolecule is expressed in Svedberg units (S), which describes its sedimentation coefficient  Using Centrifugation to Isolate Organelles © 2016 Pearson Education, Inc.  Using Centrifugation to Isolate Organelles © 2016 Pearson Education, Inc. Huber and O’Day, 2011; Huber and O’Day, 2012 Using Centrifugation to Isolate Organelles ▪ Contamination is sometimes an issue with differential centrifugation (e.g., fraction contains other organelles and cellular components) ▪ Contaminants can be removed by density gradient centrifugation ▪ Homogenized sample is placed as a thin layer on top of a gradient of solute, typically sucrose ▪ This gradient has an increasing concentration of solute – and therefore density – from the top of the tube to the bottom  Using Centrifugation to Isolate Organelles ▪ The solution is more concentrated (dense) at the bottom of the tube, and decreases in concentration gradually towards the top ▪ When centrifuged at high speed, the various organelles migrate to an equilibrium position where their density is equal to the density of the medium ▪ Particles form discrete zones or bands in the sucrose © 2016 Pearson Education, Inc.  Using Centrifugation to Isolate Organelles ▪ We can also assess the purity of the isolated sample using western blotting Senichkin et al., 2021 Recommended Readings ▪ SDS-PAGE/western blotting: Chapters 7 and 21 ▪ Protein expression vectors: Chapter 21 ▪ Fluorescent fusion proteins: Chapter 19 ▪ Centrifugation: Chapter 4