Determination Of Molecular Weight Of A Protein By Gel Filtration

3339 words - 13 pages


Purification of proteins is an essential technique that provides biochemists with the ability to obtain a sample that contains a specific protein of interest. In order to purify a protein biochemists typically obtain the specific extract, precipitate the protein using ammonium sulfate, preform gel filtration, and lastly run ionic and affinity chromatography. Understanding the use and methods of the different chromatography techniques is of crucial importance in order to successfully purify and conclude the molecular weight of an unknown sample using elution volumes of molecular weight standards.
Gel filtration or exclusion chromatography is preformed to isolate proteins of different sizes between various phases, such as solid or gel and liquid phases. If solid, the gel and beads are denoted as sorbent. Sample mixtures have two phases that they move through, a stationary and mobile phase. The stationary phase is a solid or sorbent, while the mobile phase is gas or water. The process of movement of a sample down a column is referred to as development.
Gel exclusion chromatography is an ideal method used to isolate molecules that are sensitive to harsh environments such as changes in pH or varying concentrations of metal ions. When running gel exclusion chromatography, smaller biomolecules typically penetrate the gel more and thus, take more time to elute through the column. Furthermore, larger biomolecules are too large to penetrate the gel pores and elute through the column at a faster rate. Upon coming out of the Sephedex (or out of the column) biochemists collect fractions, to further the continuation of the experiment. The diagram depicted in the figure below illustrates large molecules and small molecules eluting and the larger biomolecules making their way out of the Sephadex quicker.

Figure 1: Gel filtration columns
Selfe, S

Gel exclusion chromatography has given biochemists the opportunity to learn more about specific proteins varying characteristics such as size, shape, and solubility. Biochemists are able to utilize different equations to solve for these characteristics. The elution volume (ve ) is the volume of solvent needed for a solute to elute down a column; this is the volume of the fraction corresponding to the highest absorbance. The void volume (vo) is the volume outside of the gel particles. In order to conclude the void volume of a column one would utilize the elution volume of Blue Dextran-2000 (molecular weight of 2.00 x 106 g/mol). Each fraction collected contains 1.00 mL of the sample. For example, to determine the volume of the 2nd fraction: Volume of sample in each fraction (1 mL) x tube number (2) = 2 mL

The total volume (vt) of a packed column is calculated using the following equation:
Vt = Vi + Vg + Vo

where Vi is the internal volume or the volume inside the gel particles , Vg is the volume due to the solid walls and cross-linked internal chains of gel particles, and Vo...

Find Another Essay On Determination of Molecular Weight of a Protein by Gel Filtration

determination of sulfur by precipitation gravimetry

295 words - 2 pages sample containing an alkali sulfate is dried, weighed and dissolved in dilute HCl. Barium chloride solution is added in excess to precipitate barium sulfate, and the precipitate is digested in the hot solution. The precipitate is filtered through a paper filter which is then ignited and completely ashed. From the weight of the sample and weight of the precipitate, the

Quantitative Determination of Sulfate by Gravimetric Analysis

559 words - 2 pages thickness and width of the specimens by using a Vernier calipers.Switch on the tensile testing machine, computer and printer.Load the particular program for tensile testing of thermoplastics.Use the correct type of grips and check that the capacity of the load cell is 5KN. Make sure that the speed of testing is 50mm/min.(start from 0N)Bring down the crosshead to an appropriate level for the convenient attachment of the specimen.Key in

Molecular Basis Of Cancer

2061 words - 8 pages hepatic macrophages, the Kupffer Cells. These cells are part of the reticuloendothelial system and are phagocytic and so have an important detoxifying function. At the corners of each lobule are the portal tracts that contain branches of the hepatic artery, the portal vein and the bile ducts. Blood flows from the portal tracts towards the central hepatic vein.Liver Diseases Acute Hepatitis: Usually caused by viral infection (hep A, B, C, D, E, and

Is Whey Protein the way to go?-Research Paper on the benefits and drawbacks of using whey protein for weight training

3045 words - 12 pages whey flows. The binding is reversible via pH adjustments and the protein is in the eluted material is subsequently concentrated via ultra filtration, a membrane filtration process also used within the micro filtration process. The ion-exchange process results in chemical modifications to the proteins, with much of the nutritionally important calcium being replaced by sodium during the binding/release process."(Neville 127)Micro filtrationDuring

Comparison of Proteins in Animal Tissues By Gel Electrophoresis - Cell Biology - Research Paper

891 words - 4 pages on a white light box. The white light box allows for students to see the protein bands of the known and unknown samples. The figure represents samples known as Chicken, Beef, Fish, and the unlabeled lane is the standard provided by the professor.Figure 1: Gel of protein samples. Left to right- Molecular weight standards, beef, chicken, fish, unknown. Discussion The experiment initially wanted the samples to be heated in the

The Determination of a Topic’s Scientific Status

970 words - 4 pages example, was once thought to be a science, but later was removed as science. Although astrology uses scientific knowledge to connect astronomical phenomena to events occurring on earth, there is no method available to test the theories that stem from astrological predictions. An idea of what separates a science from a non-science, or pseudo-science, is brought upon by Karl Popper. Karl Popper’s initial thoughts for a solution to the problem of

The Theme of Determination in “A Worn Path” by Eudora Welty

677 words - 3 pages Everyday people encounter challenges that stand in their way of achieving their goals, but with sheer determination most accomplish their mission. In the short story “A Worn Path” written by Eudora Welty, Phoenix Jackson, the main character, has a mission to complete while confronting many challenges. One way to convey this idea is with the literary use of theme. The theme of a literary work is defined as the central idea, concern or message

Activation of Protein Kinases by Means of Stochastic Resonance

2173 words - 9 pages molecular motor and is based on the supposedly asymmetrical shapes of the energy landscapes of protein transitions. The particles of interest (i.e. proteins of the same kind) are initially accumulated at a position x0 where a potential minimum is found. This corresponds with the proteins having the same transition state. The resting energy of the particles at x0 is much lower than the surrounding potential barriers. The E-fields induced by the Re5

Importance of Maintaining a Healthy Body Weight

1559 words - 6 pages burns off. “A calorie is defined as a unit of energy supplied by food. A calorie is a calorie regardless of its source. Whether you're eating carbohydrates, fats, sugars, or proteins, all of them contain calories. ("Balancing Calories"). If you take in more calories than you use up it gets stored as fat. If you want to stay the same weight you have to eat the amount of calories that you use every day. If you want to lose weight you have to take in

The Importance of a Dog's Weight

1848 words - 7 pages left untreated further health problems can occur, which can eventually lead to death. Hypoglycemia, or low blood sugar, is another condition which can cause weight gain (Kay and Randolph 155). It can be cause by Addison’s disease, which is caused by “a deficiency in the secretion of hormones from the adrenal glands” (Peat). It can also be caused by a lack of protein in the diet, too much exercise, insulin overdose, and too large of a gap between

Molecular mechanisms of diabetes mellitus

1793 words - 8 pages growth factor 1 and transforming growth factor beta, which further contribute to glomerular fibrosis, with a result guiding implication of decreased availability in glomerular surface area for filtration. (David A. Antonetti Etel, 2006) Nephropathy Diabetic nephropathy is the chronic condition secondary to hyperglycemia, and possibly influenced by hypertension, depending on the individual themselves, where the hyperglycemia induces dysfunction in

Similar Essays

Determination Of Cu2+ By Titration Essay

658 words - 3 pages Determination of Cu2+ by Titration Introduction: The purpose of this lab is to determine the concentration of copper ions in an unknown solution. Complexometric titration will be used to determine the concentration. A buret will be used to deliver the complexing agent to the unknown solution. Ethylenediaminetetraacetic acid will be used as the complexing reagent. An indicator, Murexide in our case, will be used to indicate the endpoint of

A Weight Of A Nation Essay

1189 words - 5 pages The Weight of a Nation I saw so many true facts in this video that hits close to home. From the advertising to the Lower end housing. I was never obese as a child and I was always fed, but I won’t be able to say it was necessarily healthy. I pretty much ate what I wanted and if my mother was making something I did not eat she made me a separate meal, and

A Study On Topical Nanoemulsion Based Gel Of Terbinafine H Cl

1055 words - 5 pages Objective: The objective of the present study was to design topical Nanoemulsion based gel of Terbinafine HCl, a poorly soluble drug, to increase its permeability and evaluate its efficacy in an animal model. Materials and Methods: Nanoemulsions were incorporated into gels and designated as Gel-P and Gel-S respectively. Nanoemulsion gel was prepared using liquid paraffin, glycerin, and carbomer 940. Nanoemulsions were prepared by two different

Quantitative Determination Of Sulphate By Gravimetric Analysis

1614 words - 6 pages chloride was slowly added to a hot unknown sulphate solution slightly acidified with concentrated HCl. The precipitate is then filtered off by suction filtration, washed with water and dried in the oven at 150 degree Celsius for about half an hour and cooled in the desiccator, and weighed as barium sulphate and the percentage of sulphate was calculated from the weight of barium sulphate. The Molarity of sulphate= 0.004269 M and the concentration of