Lab Report #8 Protein Electrophoresis PDF

Preview

Summary

Complete Lab Report for Biochemistry on Protein Electrophoresis...

Description

Michael J. Blanco Protein Electrophoresis November 21, 2016

Abstract The molecule weight of RNase H was determined by performing odium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE separate macromolecule based on their weight. The log (kDa) vs. migration distance was graphed and a linear slope was given with a R 2 value of .97761. The molecule weight of RNase H was calculated to be 16 kDA with a percent error of 9.09%. Introduction A common method to separate protein is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS is an anionic detergent and thus the SDS contains a net negative charge that destroys most of the complex protein structures (Introduction to SDS-PAGE). The SDS disrupts the tertiary structure of the protein and thus linearized the protein (Bitesize Bio, 2016). The polyacrylamide gel restricts larger protein from migrating fast toward the anode (Introduction to SDS-PAGE). The final separation of protein is dependent on the difference in molecular mass (Introduction to SDS-PAGE). A buffer is obviously needed to conduct the current from the cathode to the anode through the gel. The discontinuous Laemmli buffer system is commonly used. The following system is set up with a stacking gel at pH 6.8 and a running gel to pH 8.8; both gels are buffered by Tris-HCl (Bitesize Bio, 2016). The stacking gel has a low concentration of acrylamide compared to the running gel (Bitesize Bio, 2016). The high concentration of the running gel is capable of delaying the movement of the proteins (Bitesize Bio, 2016). Without

the stacking gel the protein sample would stat at the top of the running gel (Bitesize Bio, 2016). Consequently, the protein in your sample would enter the running gel at different time and thus bands would be smeared (Bitesize Bio, 2016). Procedure A resolving gel and stacking gel solution was prepared for the protein electrophoresis. The resolving gel solution was prepared using 40 mL of 15% of the corresponding gel. In order to prepared the resolving gel the following supply was needed and mixed:  9.68 mL H2O  20 mL 30% Acrylamide/bis solution  10 ml 4% resolving gel buffer  300 μL of 10% Ammonium persulfate (APS)  20 μL of TEMED It is important to note, that TEMED should be added last to prevent the hardening of the solution. The 4 x resolving gel buffer was prepared with 1.5 M Tris-HCl (pH 8.8) and 0.4% SDS. The 5% stacking gel was prepared with a total volume of 20 mL. The stacking gel needed the following supply:  11.5 mL of H2O  3.3 mL of 30% Acrylamide/bis solution  5 mL of 4x stacking gel buffer  200 μL of 10% APS

 10 μL of TEMED The 4 x stacking gel buffer was prepared with .5 M M Tris-HCl (pH 6.8), 0.4% SDS. The loading sample was prepared using 3 samples from the previous lab (lab #6). From the previous lab, 15 μL of each sample was placed in a test tube, followed by 4 μL of 5x sample loading buffer, and 1 μL of 20x reducing agent. The sample was heated at 90 ℃

for 5 minutes. The sample was loaded and the

molecular weight of Ribonuclease H was determined. Results

Rainbow Ladder Distance (mm) log(kDa)

4.4 6.3 8.1 10.4 13 14.9 18.4

1.579783597 1.491361694 1.380211242 1.230448921 1.079181246 0.929418926 0.544068044

Log(kDa) vs Distance 2 Log(kDa)

1.5

f(x) = − 0.07 x + 1.95 R² = 0.98

1 0.5 0 2

4

6

8

10 12 14 16 18 20

Distance (cm)

Calculation of molecular weight of RNase H: y=−0.0716 x +1.9487

y=− 0.0716(10.4 cm )+ 1.9487 y=1.20406 y 1.20406 MW =10 =10 =16 kDa Percent Error: Distance Calculated (cm) kDa Original Flowthrough Washing Flowthrough Elution Flowthrough

13

10.42077

13

10.42077

10.4

15.9977

17.6 −16 ×100=9.09 % Error 17.6

Discussion

The purpose of the experiment was to become familiar with SDSpolyacrylamide gel electrophoresis (SDS-PAGE) of protein, as well as, determine the molecular weight of RNase H. The SDS-PAGE was prepared by creating resolving gel and stacking gel solution. The sample was prepared and loaded from the prior lab (lab #6). The molecular weight of RNase H by finding the linear slope of the graph Log(kDa) vs. Distance. The equation given by the slope is as follow: y=−0.0716 x +1.9487 The eluting buffer’s migration distance is the x value. The eluting buffer contains our wanted protein. The molecular weight calculated for the eluting buffer is 16 kDa compared to the literature value of 17.6 (Ribonuclease H from Escherichia coli (R6501) ). The percent error calculated is 9.09%, which is quite reasonable. Possible sources of error that developed throughout the experiment are the incubation time was less then required. The incubator was slow in heating and we did not have sufficient time to incubate our sample after the class.

References

How SDS-PAGE works - Bitesize Bio. (2016). Retrieved November 21, 2016, from http://bitesizebio.com/580/how-sds-page-works/ Introduction to SDS-PAGE. (n.d.). Retrieved November 21, 2016, from http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html Ribonuclease H from Escherichia coli (R6501) - Product ... (n.d.). Retrieved November 21, 2016, from https://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Product_Information_Sheet/1/r6501pis.pdf...