Lab 5 Protein Analysis- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) PDF

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Lab 5 - full lab report...

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I. II.

Title: Protein Analysis- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Date: 02/12/19

III.

Lab Partners: Isaiah Muniz, Kimia Rezaeizadeh, and Shadi Azin

IV.

Purpose: The primary purpose of this experiment is to gain hands-on experience in preparing protein samples for SDS-PAGE, loading a protein gel, running SDS-PAGE, and measuring the relationship between migration distance and protein molecular weight.

V.

Methods:

VI.

Results:

Figure 1.1 Gel Electrophoresis 15 minutes after the run prior to the staining.

Figure 1.2 Gel Electrophoresis 15 minutes after the run prior to the staining corresponding to group 3.

Figure 2.1 Gel Electrophoresis stained with Coomassie blue

Figure 2.2. Coomassie-stained gel electrophoresis with migration distances(cm).

Table 1. Migration Distances of Protein Bands on SDS-PAGE

Figure 3. A photo of the Coomassie –stained gel with the lanes labeled and the band sizes (in kDa)

Table 2. Migration distance, known molecular weight, and log of molecular weight of protein standard. Mig. Distance (cm)

Log of MW (kDa)

MW (kDa)

0.6

2.40

250

0.8

2.18

150

1.3

2.0

100

1.5

1.88

75

2.1

1.70

50

2.8

1.57

37

3.7

1.40

25

4.3

1.30

20

5.2

1.18

15

5.9

1

10

Figure 4. Standard curve of the standard protein; migration of the log of MW versus migration distance.

Table 3: Molecular Weight and the estimated number of amino acids of Samples Sample

Size in kDa (Based on gel calculations)

Estimated # of Amino acids

Size in kDa

Ovalbumin

46.3

421

42.7, 44.3

Commercial ovalbumin

48.9

445

42.7, 44.3

BSA

67.7

616

65

Antibody Heavy Chain

68.4

622

50

Antibody Light Chain 28.4

258

25

Sample Calculations: ● Size in kDa Y =− 0.2355x + 2.3251 Y =− 0.2355 (2.8) + 2.351 = 1.692 S ize in kDa = 10 1.692 = 46.3 kDa ● Estimated # of Amino acids: 46300 Da 110 Da/Amino acid

= 421 Amino acids

VII.

Discussion: 1. What was the purpose of heating the sample in a heating block prior to loading it onto the gel? Firstly, the heat denatures the globular structure of the molecules (weak bondings like hydrogen bonds) so it facilitate the migration of the pieces of DNA or protein down the gel by their size not their globular 3D structure. Secondly,SDS binds to the denatured protein. Once denatured, the SDS binds along the amino acid chain and both neutralizes the charge and prevents the protein from re-folding. This is why it only needs to be boiled  biomolecules rupture at high temperatures except stable nucleic once. Lastly,  all acids,therefore to remove or deactivate the impurities(other biomolecules of the cell) from the sample of DNA, the sample is boiled for gel electrophoresis. As this process is based on the principle of relative specific gravity (or density) therefore removal or deactivation of other molecules or compounds (are usually heavy) is very necessary for expected results. 2. In the lane containing the antibody sample, why are 2 bands present?(hint: look up the structure of a general antibody molecule.) Antibodies are glycoproteins belonging to the immunoglobulin family. They are heavy globular plasma proteins.They  have sugar chains (glycans) added to amino  acid residues. In other words, antibodies are glycoproteins . The Ig monomer is a "Y"-shaped molecule that consists of four polypeptide chains. They are typically made of basic structural units—each with two large heavy chains and two small light chains. Heavy chains contain about 400-500 amino acids,whereas the approximate length of a light chain is 211 to 217 amino acids. 3. What is the purpose of: a. bromophenol blue- A tracking dye for protein electrophoresis in polyacrylamide gels.Bromophenol blue is used as tracking dye in electrophoresis. It has a slight negative charge and will migrate the same direction as the protein, allowing the user to monitor the progress of molecules moving through the gel. b. protein standard- To determine the molecular weight of an unknown protein, a series of molecular weight standards with known values are run in parallel with the unknown protein called standard protein c. glycerol- Glycerol is used both in sample preparation and gel formation for polyacrylamide gel electrophoresis. Glycerol increases the density of a sample so that the sample will layer at the bottom of a gel’s sample well. Glycerol is also used to aid in casting gradient gels and as a protein stabilizer and storage buffer component.

d. SDS- SDS (sodium dodecyl sulfate) is used to denature the proteins. It’s an anionic detergent, meaning that when dissolved its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field. 4. How do your calculated MW's compare with the known MW of each protein? Considering experimental error in the lab we did very good on calculating the molecular weight of each protein. While we had our molecular weight only 2.7 kD’s off the known weight of BSA we had a bigger error in finding the molecular weight of the Antibody Heavy Chain which we got 18.5 kD’s off. 5. Refer to the Egg Whites Lab and recall the actual protein concentration of ovalbumin you had. Show your calculations for preparing the 5 μg ovalbumin sample, including the volume of 2x FSB (for a final 1x FSB). How does your student-prepared ovalbumin differ from the commercial ovalbumin? Why? Calculation for commercial Ovalbumin: (2X)(V 1 ) x (10 µL )(1X) V

1

= 5µL

5 µL of 1 µg/µL Ovalbumin + 5 µL of 2x FSB = 10 µL total Calculation for the concentration of experimental ovalbumin: (46 µg/µL)(V ₁) = (1µg /µL)(100µL) (V ₁) = ( 100/46) µL of ovalbumin (V ₁) = 2.2 µL of ovalbumin + 97.8 µL of water Well looking at the SDS-PAGE results our second mark is visible but not as clear or thick as the commercial ovalbumin. The likelihood of matching exactly as the commercial ovalbumin is tough but possible, our concentration was calculated correctly but there may have been other issues with our experiment that we may have caused. Possibly diluting too much of our student-prepared ovalbumin caused this issue or possibly making small pipetting errors that caused the difference in the appearance of our mark....