Lab Protocols

Background: Before you start any type of lab work, it is a good idea to check the accuracy and precision of the micropipetters that you plan to use. A very simple way to do this is by weighing the volume of water actually transferred by a micropipetter at a given setting.   Water has a density of 0.9986 g/mL at room temperature, so you can use the mass of the water transferred to determine the accuracy of your pipetter (as long as your balance is reliable of course....), and by repeating the test several times, you can determine the precision of the micropetter as well.. Procedure : 1. Using a balance capable of reading in milligrams or lower, tare a plastic weighing boat on the pan of the balance. 2. Set the micropipetter to its maximum capacity and carefully transfer that volume of distilled water to the weighing boat. 3. Repeat the operation at least four times, each time recording  the weight of distilled water transferred. 4. Set the micropipetter ...

 Total Protein Staining  and Immunostaining  are procedures applicable to proteins bound to nitrocellulose membranes.  The proteins may be applied to a nitrocellulose membrane by electrophoretic transfer, dot or slot blotting, tissue printing, etc.  Usually the nitrocellulose is stained first for total protein and photographed (Total Protein Staining ). The stain is then removed and the nitrocellulose membrane restained immunologically, using antibodies specific of the protein of interest (Immunostaining). Total Protein Staining  A. Reagents and Solutions     * CPTS Staining Solution - (0.5 mg/mL CPTS)  Dissolve 50 mg of 3,4',4 ²,4-copper phthalocyanine tetrasulfonic acid, tetrasodium salt (CPTS) in 100 mL of 12 mM HCl.  The solution is stable at room temperature indefinitely.     * Wash Solution - (12 mM HCl, pH 2)  Dilute 1.0 mL conc. HCl in 999 mL dH2O. This solution is stable forever at room temperatur...

The molecular weight  standards used in the  SDS-polyacrylamide gel electrophoresis and Western blotting were, "Color Markers, Wide Range" (Sigma).   The dye attachment increases the apparent molecular weights of the modified proteins.  The column headed "Apparent Molecular Weight (in Daltons)" is the estimate provided by Sigma for their apparent molecular weights when used as standards in SDS-PAGE.  My personal experience is that the apparent molecular weights actually determined by SDS-PAGE are usually somewhere between the actual protein molecular weights and the estimates from Sigma.  Life is never simple, is it?   Protein Molecular Weight  (in Daltons)     Color  Apparent Molecular Weight (in Daltons) Myosin, Rabbit Muscle 205,000 Blue 225,000 beta-Galactosidase ( E.coli ) 116,000 Turquoise 128,000 Albumin, Bovine Serum 66,000 Pink 73,000 Ovalb...

 I. BACKGROUND & PURPOSE Gel electrophoresis is a useful method to separate and/or identify proteins and nucleic acids. In SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins are separated largely on the basis of polypeptide length, and so their molecular weight can also be estimated. SDS does however denature the protein, so activity stains cannot be used to identify particular enzymes. Described below is the protocol for preparing and using Laemmli discontinuous gels. In this system, two sequential gels are actually used; the top gel, called the stacking gel, is slightly acidic (pH 6.8) and has a low (5.5%) acrylamide concentration to make a porous gel. Under these conditions proteins separate poorly but form thin, sharply defined bands. The lower gel, called the separating, or resolving gel, is more basic (pH 8.8), and has a higher polyacrylamide content (in our case, 12%), which causes the gel to have narrower channels or pores. As a protein, concen...

I- BACKGROUND AND PURPOSE a -Amylase ( a -1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) is an enzyme that degrades starch, first to oligosaccharides and then in turn to maltose and glucose, by hydrolyzing  a -1,4-glucan bonds.  In digestion, the role of a -amylase is primarily the first reaction of this process, generating oligosaccharides that are then hydrolyzed by other enzymes.     a -Amylase    a -Amylase Starch    --->  Oligosaccharides    --->  Maltose + Glucose In vitro , a- amylase is also able to hydrolyze the  a -1,4 linkages in glycogen, but has no activity on the   a -1,6 linkages responsible for the more highly branched structure of glycogen.  These branched structures also reduce the activity of a -amyl...

Purpose:     Identify which samples contain real maple syrup, and which contain fake maple syrup, and determine which carbohydrates the imposters contain.     Qualitative Carbohydrate Assays   (It is useful to run both negative and positive controls at the same time as the sample.)   Molisch Test (Carbohydrates)     Prepare Molisch's reagent by dissolving 0.5 g reagent grade a -naphthol in10 mL of 95% ethanol.  Store the reagent, protected from light, at room temperature.  To test for carbohydrates, add 0.02 mL of the reagent to 1 mL of 0.1% carbohydrate (1 mg/mL) solution in a small test tube.  After mixing, tilt the tube and carefully add without mixing , 0.5 mL of concentrated sulfuric acid by pouring it down the side of the tube. (Use a glass Pasteur pipette to add the H 2 SO 4 : do not use a mechanical pipettor with concentrated acids.)  A red-violet layer at the interface between the acid (bottom) and...

Purification protocol  Background & Purpose Cytochrome c, a bright orange pink, iron containing protein which functions as an electron shuttle in the mitochondrial electron transport path, can be purified from beef heart muscle by selective precipitation, ion exchange and/or gel filtration column chromatography. Cytochrome c is a convenient protein to isolate, because it is quite stable and because its bright color makes it easy to follow during the purification. The purification protocol that we will use is based on the fact that cytochrome c has several positively charged groups, giving it a pI of around 10. Thus, it is normally bound to the inner membrane of mitochondria by ionic attraction to the negative charges of the phospholipids on the membrane. The tissue and mitochondria are first broken up by homogenization in a blender at low pH, in an aluminum sulfate solution. The positively charged aluminum ions can displace the cytochrome c from the membrane by binding to ...

GEL FILTRATION AND ION EXCHANGE CHROMATOGRAPHY Background Ion exchange chromatography Ion exchange chromatography separates substances on the basis of their charge. There are two general classes of ion exchange media or resins; anion-exchange media, which have positively charged groups attached to the media, and bind to anionic (negatively charged) compounds, and cation-exchange media, which have negatively charged groups attached to the media, and bind to cationic compounds. Amberlite CG-50 is a cation-exchange resin, i.e., it has covalently attached carboxylate groups which, at neutral pH, are charged-balanced by associated sodium ions. Proteins with significant regions of opposing charge will displace the sodium and bind to this column material by ionic attraction. The initial protein binding is usually done in a solution of low ionic strength where the protein, in this case cytochrome c, can displace the sodium ions, and bind to the ...

To determine the amount and purity of the cytochrome c you have isolated, two independent methods are required. First, the visible absorbance difference spectra, which is dependent on the difference in absorbance between reduced and oxidized heme iron, is used to determine the cytochrome c concentration of the solution, in units of millimoles/Liter (mM). From this, using the volume of the solution and the molecular weight of cytochrome c , (12,327 g/mol) we can calculate the amount of cytochrome c (in grams). Then, by using the Bradford protein assay we can determine the total concentration of protein in the solution, in units of milligrams/milliliter (mg/mL) and using the volume of the solution, the total amount of protein (in grams). By comparing the amount of cytochrome c (in grams) and the total amount of protein (in grams) we can determine the purity of our preparation. These two methods should be used to also determine the concentration of cytoch...

Buffer pK a Neutral form HCl salt Na + salt H 3 PO 4 / NaH 2 PO 4 (pK a1 ) 2.12 98.0 - 120.0 Glycine (pK a1 ) 2.34 75.07 111.5 - Citric acid(pK a1 ) 3.13 Acetic acid 4.75 60.05 - 82.0 Citric acid(pK a2 ) 4.76 192.1 - 294.1 MES 6.15 195.2 - 217.2 Cacodylic acid 6.27 H 2 CO 3 / NaHCO 3 (pK a1 ) 6.37 62.01 - 84.01 Citric acid(pK a3 ) 6.40 Bis-Tris 6.50 209.2 245.7 - ADA 6.60 190.2 - 212.1 Bis-Tris Propane (pK a1 ) 6.80 282.4 318.9 - PIPES 6.80 302.4 - 325.3 ACES 6.90 182.2 218.7 - Imidazole 7.00 68.1 104.5 - BES 7.15 213.2 - 235.2 MOPS 7.20 ...