Enzyme Kinetics


 Introduction: 

SDS-PAGE is a electrophoresis method with using Sodium Dodecyl Sulfate as a denaturating agent for proteins. Polyacrylamide gel provide porous structure to molecular weight separation of proteins or nucleic acids. SDS is used when to break single polypeptide chain. However, multimeric polypeptides includes disulfide bonds that SDS not sufficient to breake them. Instead of using SDS sample is treated with β-mercaptoethanol to break disulfide bonds. In order to seperate proteins, electrophoresis apparatus were developed. At the cathode side samples are placed to wells, and current is applied samples moves to anode side. (1*) 

Polyacrylamid gel has two different gel layer that are stacking gel and resolving gel. 

Stacking gel is composed from %5 acrylamide-bisacylamide.In this gel pH level is 6.8 that glycine is zwitterion form, so immobilized in gel because of that negatively charged proteins moves between clorine and glycine. This mechanism called as sandwich mechanism. By this way samples are reach resolving gel at the same time. When proteins reach the resolving gel, they moves according their molecular weight. In this gel pH level is 8.8 and glycine is at negative form, so it can freely mobilize.Protein’s movement would not be dependent sandwich mechanism anymore. 

To prepare polyacrylamide gel APS and TEMED initates the reaction and other catalyze the synthesis respectively.TRIS/HCI buffer used for stabilize the pH and provides CI ions.Glycerol is used to perticipates the samples into wells.Bromophenol is used to trace the migration of proteins.(2*) 

In this laboratory previously purified His-TAG proteins and samples that obtained form size exclusion were run into gel to seperate due to their size.   

Materials and Methods: 

Apparatus were prepared. Two glass were clipped with stand. To understand water leakage, distilled water were pour the space between those glasses, and system is up side down. 

Required amounts of gel preparations were indicated below 

%15 Resolving Gel  Component volumes (ml) 
Solution Components  10 ml 
H20  2.3 
30%acrylamide mix  5.0 
1.5M Tris (pH 8.8)  2.5 
10% SDS  0.1 
10% ammonium persulfate  0.1 
TEMED  0.004 

 

%5 Stacking Gel  Component volumes (ml) 
Solution Components (%15)  10ml 
H20  6.8 
30%acrylamide mix  1.7 
1.0 M Tris (pH 6.8)  1.25 
10% SDS  0.1 
10% ammonium persulfate  0.1 
TEMED  0.01 


Discussion:
 

In first gel different bands were seen in different wells. Cell lysate were expected to has greater protein than others, therefore much more bands in second well was seen. Also other protein and wash contained proteins different than protein of interest. In other protein sampe and wash sample mostly one particular protein was removed. This band seen also in elution sample, so mostly removed protein could be protein of interest. When we reject that assumption we have to conclude that elution sample would not be includes correct protein.  

Gel that were loaded with oval albumin and lysosyzme had to showed single band with that has same sample. Lysosyzme should be seen single band and oval albumin should be seen another single band. DNA ladder that was in Gel 2 did not provide clear information to these computation. Gel 3 and Gel 4 exhibits different bands for those samples. Even though DNA ladder not clear there are distinct seperation between two bands. C11,C12,C13 must included lysosyzme because it reaches the gel’s at faster, it shows that it has lower molecular weight.*3 

When, lysosyzme has smaller molecular weight than oval albumin, this result expected.   

When size exclusion use bead , polyacrylamide gel uses porous structure. Main difference between them is one of them retard the mobility of the smaller molecule with their sieve like structure that is size exclusion’s bead model. Polyacrylamide gel creates porous structure and matrix, because of that molecules of sample could not leave gel and larger molecules has slower movement than smaller molecules. 

In this experiment, obtained proteins’s control could be done by SDS-PAGE. SDS can also purify sample , because of that it is important. Gel preparation and main idea of the electrophoresis was understood in this laboratory. 

References: 

  1. http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html 
  1. https://ww2.chemistry.gatech.edu/~lw26/course_Information/4581/techniques/gel_elect/page_protein.html 
  1. https://www.sigmaaldrich.com/catalog/product/sigma/a5503?lang=en&region=TR 

 

 

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