My Practical Reports: SDS-PAGE

 

sds page lab report

Nov 09,  · View Lab Report - SDS PAGE lab report from CHEMISTRY at Baylor University. Experiment #9: Protein Identification by SDS-PAGE Shaina Bail %(39). Bio 6 – SDS-PAGE Lab Objectives Upon completion of this laboratory you will understand how to load and run protein samples on an SDS-polyacrylamide gel, stain the gel, and analyze the resulting bands of protein on the gel to estimate the. Lab Report - What to Turn In for Lab Report 4 Prepare report run, stain and destain an SDS-PAGE during the 3 hour lab. So, your lab this week is to analyze the gels of your samples from the GOT purification. As in the Native PAGE gel Documents Similar To protein electrophoresis lab. Carousel Previous Carousel Next. Protein Assay by the 5/5(1).


protein electrophoresis lab | Polyacrylamide Gel Electrophoresis | Gel Electrophoresis


Why do you think one is better than the other in resolution…What are the advantages of each gel method? During denaturation by boiling, all disulfide bonds in the protein are reduced with 2-mercaptoethanol some times called beta-mercaptoethanol and the protein subunits ie polypeptide chains are uniformly bound with the detergent sodium dodecyl sulfate SDSwhich has the structure CH3 CH2 SO3- and the sodium is just a counter ion.

The detergent gives the polypeptide a uniform negative charge and binds in proportion to size of the subunit, sds page lab report.

As a consequence, all the protein subunits in a mixture of proteins have the same charge density and will migrate in an electric field with the same mobility. Thus, an SDS-PAGE gel can be calibrated with proteins of known subunit size or molecular mass - which is called Mr and so the molecular mass of an sds page lab report polypeptide can be determined. This is best done by preparing a plot of the log Mr of the protein bands representing the standard proteins versus electrophoretic mobility of the protein bands as compared to the dye front called relative mobility.

Or in some cases, you may have been purifying an enzyme from a new source and know the Mr of its subunit from another source for example, the enzyme you are purifying is from spinach leaves and the enzyme has already been purified from corn leaves and its Mr determined. Native protein is unfolded by heating in the presence of a disulfide bond reducing agent and SDS. Many proteins contain disulfide bonds Cys-S-S-Cys joining the polypeptide backbone and to remove these bonds, a disulfide reducing agent like beta-mercaptoethanol is used.

Sds page lab report heating the protein would normally precipitate, but the SDS binds to the backbone and provides a negative charge to make the denatured protein soluble.

The hydrophobic tail ie the dodecyl part of SDS binds to the hydrophobic backbone of the protein, and the ionic sulfate group projects out into solution making the denatured protein soluble.

Binding of SDS to the protein is in proportion to protein size. Large polypeptides bind more SDS than small polypeptides. So proteins end up with negative charge in relation to their size.

During electrophoresis, large proteins move less distance in the gel than small proteins. So bigger polypeptides move slower during the electrophoresis, sds page lab report.

The protein mixtures obtained at each step in a typical purification are shown with the pure enzyme in lane 4 of the gel shown.

The Purification steps and the lane labels on the gel are: 1. Crude Extract -- total mixture of all proteins at the start of the purification. After Ion Exchange Chromatography -- containing enzyme activity of sds page lab report and a mixture of proteins. After Gel Filtration Chromatography -- containing enzyme activity of interest and sds page lab report mixture of proteins. After Affinity Chromatography -- containing enzyme activity of interest and a single protein.

Standard Proteins of known molecular weight for their subunits. Electrophoretic mobility means how far the protein moved in the gel during electrophoresis. The standard proteins have known subunit molecular weights. Using the plot, the MW of the unknown pure protein is determined. Only pure proteins can be used for estimating subunit MW, since the sds page lab report of contaminating proteins would lead to confusion.

So, your lab this week is to analyze the gels of your samples from the GOT purification. The gel will also have a lane with a mixture of standard proteins for calibrating the gel you will be given a handout in lab which will identify the Mr of the standard proteins found on your gel. You should make a drawing of sds page lab report gel, which shows the intensity of the stained protein bands and sds page lab report the electrophoretic mobility of all the bands in the lane with the standards, the lane with your CMC fraction and the lane with the Sigma standard, sds page lab report.

With this information, make a standard curve for the calibration of the gel using the given Mr values for the standard proteins and estimate the Mr of all bands in your CMC fraction as well as the Sigma GOT.

Since your CMC fraction will contain more than one band and as it turns out so does the Sigma GOT, try to use the staining intensity of the bands to decide which is the major protein and assume that this is the subunit of GOT.

The data to do this is provided here. You should treated this data as if you collected it in the lab running the FPLC system during your lab this week. I carried out the experiment for you and the data I collected are below.

The standard proteins used for calibration and their native MW are:. Vo void volume of the column is equal to the time it takes Blue Dextran to elute from the column since it has a MW of more than 1 million. Ve is the elution volume of the various standard proteins and GOT. Measure these values from the data on the chromatograms shown below. Volumes are given in minutes, which is equivalent to volume since the FPLC gel filtration column is pumped at a constant flow rate of 0.

The total volume of the column is 24 ml so things eluting at 45 to 50 min are very small. A typical calibration curve obtained with the Sigma MW-GF kit used for this experiment is shown below. If sds page lab report do not have Semi-Log paper, then take log of MW of standards and plot on linear graph paper as shown above. By quaternary structure, I mean is GOT a monomer, dimer, or tetramer or what, according to your data.

This is done by dividing the Native MW obtained from the gel filtration estimate by the subunit Mr or MW obtained from the SDS-PAGE analysis and rounding to the nearest whole number since a protein can not contain a fraction of a subunit. Campbell,, All Rights Reserved; wcampbel mtu, sds page lab report. Read Free For 30 Days. Uploaded by Marie St. Flag for inappropriate content. Related titles. Carousel Previous Carousel Next. Sds-polyacrylamide Gel Electrophoresis Introduction.

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sds page lab report

 

Nov 09,  · View Lab Report - SDS PAGE lab report from CHEMISTRY at Baylor University. Experiment #9: Protein Identification by SDS-PAGE Shaina Bail %(39). SDS-PAGE and Western Blotting Lab report (extensive methods section) Essay. Abstract. This experiment made use of the Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis technique to plot a curve displaying the electrophoretic mobilities of 7 proteins against the known molecular dobyermansa.ga: Alex. Lab Report - What to Turn In for Lab Report 4 Prepare report run, stain and destain an SDS-PAGE during the 3 hour lab. So, your lab this week is to analyze the gels of your samples from the GOT purification. As in the Native PAGE gel Documents Similar To protein electrophoresis lab. Carousel Previous Carousel Next. Protein Assay by the 5/5(1).