Bard College, BIO 141

Excerpt: ... ually or by spectroscopy and is an indication of the presence of human anti-HIV antibodies in the serum of the individual being tested, and hence of exposure to the virus. In a negative reaction there will be no color produced, because the subject's serum contained no antibodies to HIV p24, so there is nothing for the rabbit anti-human IgG-HRP to bind to. Sunday, August 31, 2008 Page 73 9:04:59 AM BIOLOGY 141: SUBCELLULAR BIOLOGY - LABORATORY MANUAL EXERCISE 10: ENZYME-LINKED IMMUNOADSORBENT ASSAY (ELISA) Figure 11-1. Relationship of target antigen (HIV p24), the subject's antibody (human anti-p24), and the detecting rabbit anti-human IgG-HRP in a positive enzyme-linked immunoadsorbent assay for exposure to the virus. In today's laboratory you will use this procedure to assay for the presence of rabbit anti- bovine serum albumin in three unknown sera. You will therefore be using bovine serum albumin to coat the wells of the microtiter plates and goat anti-rabbit IgG-HRP for visual detection. In additio ...

UCSC, BIO 100

Excerpt: ... Enzyme Extraction and Sample Preparation Today you will prepare a sample of the enzyme LACTASE. Later you will determine the protein concentration in your sample and measure the enzyme activity in a standard assay. Start with one of the over-the counter dietary aids that contain lactase, such as "Dairy Digestive", "Lactaid", or "Dairy Relief". Read carefully, and record, the information given on the package, particularly noting any information about the ingredients. These products are sold in tablet form, and contain substances other than LACTASE. The protein determination methods and the enzyme assay involve spectrophotometry. Therefore the enzyme extract must be "clarified". (i.e. all insoluble material removed). Clarification is done by centrifugation, followed by membrane filtration. Some of your clarified extract is set aside for protein determination. The rest, destined for enzyme activity studies, is stabilized by the addition of BSA ( bovine serum albumin ), glycerol, and DTT (dithiothreitol) and kept i ...

Michigan State University, BIO 111L

Excerpt: ... BS111L Lab 5: Protein Fingerprinting Notebook Summary Written by Tyler Syring Lab Partner: Devan Irving Objective We chose to test scenario 2. This, in general, is to test if the salmon picked up at the grocery store for dinner is actually salmon, or just a trout that's muscle has been stained pink to resemble salmon. Results Protein's Myosin Myosin Heavy Betagalactosidase Bovine serum albumin Actin Tropomyosin Carbonic anhydrase Soybean trypsin inhibitor Myosin light chain 1 Myosin light chain 2 Myosin Light Chain 3 Lysozyme Aprotonin Actin Shark Myosin + + + + + Mom's Salmon + + + + + + Trout + + + + + + Salmon + + + + + + Kaleidoscope + + + + + + + + - + + - + + + + + + + + + + + + + Samples Mom's Salmon vs. Salmon Mom's Salmon vs. Trout Mom's Salmon vs. Shark Proteins in Common 6 11 5 Total Proteins 13 13 13 % Commonality 46.0% 85.0% 38% As you can see from the table above, the two samples that most resemble each other by protein characteristics happen t ...

USC, BISC 220

Excerpt: ... Chapter 8: ELISA technique Specifications Coating Buffer: 0.1% Sodium Bicarbonate, pH 8.6. Coated antigen: Salmonella typhimurium Washing buffer: 0.01M Phosphate Buffered Saline pH 7.4 with Tween 20 (.05%) Blocking buffer: 0.01M Phosphate Buffered Saline pH 7.4 with 1% Bovine serum albumin (BSA) Primary antibody: Abcam ab35156 rabbit polyclonal IgG (100x dilution in blocking buffer) Enzyme-labeled secondary antibody: Sigma A3687 polyclonal goat anti-rabbit IgG, alkaline phosphatase (AP) conjugated (1000x dilution in blocking buffer) Substrate: BCIP/NBT (alkaline phosphatase; chromogenic color change) BCIP: 5-bromo-4-chloro-3-indolyl phosphate NBT: p-nitroblue tetrazolium chloride. Results Just as the lab manual describes, well 12 (your control) should be regarded as colorless (though color can evolve slowly) because there was absolutely no patient serum added. Since the "patient serum" contains antibody, well 1-11 would show blue color gradually changing from dark to light, to colorless. Note These are fro ...

U. Houston, WHO CARES 23948

Excerpt: ... d interacts w/ the products of the response o Aka immunogen A substance capable of eliciting an immune response. All immunogens are antigens, but some antigens (ex haptens) are not immunogens o Immunogenicity The capacity of a substance to induce an immune response under a given set of conditions o Antigenicity The ability to combine specifically w/ the final products of the above responses Ex. Antibodies and/or cell-surface receptors Incomplete antigen o Cant induce immune response by itself; can function as an antigen o Some small molecules called haptens, are antigenic but incapable, by themselves, of inducing a specific immune response o Haptens Lack immunogenicity - - - 4 - Karl Landsteiner chemically defined system for studying the binding of and individual antibody to a unique epitope on a complex protein antigen Small organic molecules that are antigenic but not immunogenic Chemical coupling of a hapten to a large protein (like Bovine serum albumin ), called a carrier, yields an immunogenic h ...

UC Riverside, WHW 3234

Excerpt: ... RESTRICTION DIGESTION OF DNA wisdom from Howard Judelson Read the "rules" at the end of this document! Setting up the digest 1. Combine the following in a microfuge tube in order (this is for a 20 l digest; this can be scaled up or down): 2 l 10x Buffer 13 l water (to 20 l final volume) 0.2 l 100X BSA (optional*) 2 l DNA (0.5-3 g depending on application) XX l Enzyme* *In general, aim for about 10-fold overdigestion in a 2 hour digest. By definition, 1 unit of enzyme cuts 1 g of DNA per hour. So if you are cutting 1 g of DNA for two hours, this means a 10-fold excess would be 5 units of enzyme. Most digests use about 0.5 l of enzyme per reaction, which is usually 5 units. * In nearly all cases, enzyme performance is enhanced by the presence of acetylated bovine serum albumin (BSA) in the reaction. Sometimes the effect is minor, sometimes major. Rather than try to remember which enzymes require BSA, I always add it! 2. Gently flick the tubes (don't vortex, this denatures proteins) and spin down in ...

CSU San Bernardino, C 436

Excerpt: ... hate buffer. Assay each of these solutions as well as the undiluted sample using the assay protocol described below. To determine which samples are giving a response (absorbance at 400 nm) in the linear range, one can divide the absorbance by the volume of the original enzyme solution used (1x10 -2 mL, 2x10-3 mL, etc.) Those samples giving approximately the same absorbance/mL values are yielding a linear response. This value may then be used to calculate the -glucosidase activity of the sample. Standard Assay Protocol for a-Glucosidase Set up plastic tubes for the assay and include one for a blank. To each tube add 0.90 mL of 0.10 M sodium phosphate, pH 6.4, containing 1.5 mg/mL of bovine serum albumin . Also add 0.10 mL of 2 x 10-2 M p-nitrophenyl- -D-glucopyranoside (a-PNPG). Vortex all tubes and place them in a rack in the 70 oC water bath. Prewarm for 5 min , then add 10 L of the enzyme and start timing the reaction. Use a Eppendorf or similar pipet to add the enzyme. Vortex each tube immediately after a ...

SD State, MICR 422

Excerpt: ... cells + Antigen (peptide) Effector T + memory T cells (CTL, T H) 2 When is an antigen NOT an immunogen? Haptens are small molecules, usually organic, which may bind to antibody or TcR, but will not by themselves induce an immune response To generate antibody against haptens, they must be conjugated to a complex protein carrier (e.g. Bovine Serum Albumin ). What Influences Immunogenicity? Humoral Immunity - Proteins, polysaccharides are good immunogens Lipids, nucleic acids generally poor immunogens (with some exceptions) Cell-mediated Immunity - Proteins, SOME lipids and glycolipids(nontraditional using CD1). Immunogenicity cont'd. Type of Immunogen "Foreignness" Size Chemical Composition Susceptibility to Ag processing Host Factors Genotype (MHC linkages) Dosage/Route of Administration Adjuvants 3 Foreignness of the Antigen Immunogens must be NON-SELF i.e. must NOT have been seen during "education" in the bone marrow or thymus This could be due to: Phylogenetic distance - mice, rabbits, and goat ...

University of Illinois, Urbana Champaign, PHYS 552

Excerpt: ... we used in Lab 5, and bovine serum albumin (BSA), a protein used as a carrier of nonwater soluble molecules in the bloodstream. Both proteins contain the fluorescent amino acid tryptophan, whose fluorescence we will be measuring. 1. Measure the anisotropy for each of the proteins. (See page 6 for general directions). a. Tryptophan settings: Excitation at 280nm, polarizer Emission at 335 nm, number of iterations = 40. b. Detect G-Factor once for each sample. Analysis We will use the Perrin equation and the equation relating the rotational constant to the MW of the protein: = V RT = RT MW( + h) where is the specific volume (~0.73mL/g) and h the hydration of the protein (~0.23 g water/ g protein) You will also need the values r0 = 0.26 for tryptophan, = 2 (average for proteins), MW of BSA = 66,400 g/mol, MW of Lysozyme = 14,300 g/mol. Q7) First assume that the fluorescence lifetimes of the tryptophans are the same in the two proteins and derive a relationship relating the anisotropies and ...

Michigan State University, BIO BS 111

Excerpt: ... L 10 L 10 L 10 L 10 L 10 L Sample Kaleidoscope Mom's Salmon Brown Trout Known Salmon Scallop Actin/Myosin The difficulty with this assignment is the possible sources of error. Due to the high subjective nature in determining whether a line is, indeed, a protein band, there are varying interpretations from person to person. The other two members of my group and I looked at the gel results very closely and, using actin/myosin and kaleidoscope standards, came up with the interpretation found in Figure 2. Protein Present Myosin Myosin Heavy Chain BetaGalactosidase Actin Myosin Scallops Salmon Trout Mom's Salmon Kaleidoscope + Size 198.3 kD 190.6 kD Distance 12 mm 12 mm 14mm 15 mm + + + + + + + + + 131 kD + Bovine Serum Albumin + 82.7 kD 17mm 19 mm 21 mm + + Actin + + + + + + + + + + + + + + + 24 kD + 31.3 kD + 40.4 kD 35 kD 43 kD + + + + 23 mm 24 mm 25 mm 26 mm 27 mm 28 mm 29 mm 32 mm 34 mm 36 mm Carbonic Anhydrase Trop ...