Determination of Molecular Weight by SDS-Page
Introduction: The purpose of this experiment was to identify an unknown protein sample by SDS-Page. SDS-page is a shortened name for sodium dodecyl sulfate polyacrylamide gel that separated proteins based on the size of the molecules using negatively charged detergents (Huang 2016). The size of the molecule determined the distance traveled by the protein as larger proteins traversed shorter distance and visa versa. Due to size of the proteins being the main determination factor, it was essential to establish the same charge to mass ratio; specific reagents were used to meet this goal. β-Mercaptoethonol was used as a reducing agent to disrupt the tertiary structure and disconnect …show more content…
The samples were placed for five minutes in a 95 C heating block. After the instructor loaded the standard into the gel, members loaded their labeled protein samples into the appropriate electrophoresis wells. The cover of the apparatus was attached and the associated colored cables. The apparatus was slowly turned on to a voltage of 90 V and allowed to run. The standard loaded by the instructor was monitored as it was dyed and was used to measure the completion of the run. As the gel was ran, ImageJ software was explored on a sample picture of a ran gel to analyze the distance traveled. After about 40 minutes, the gel was removed and rinsed with deionized water for three minutes, and about 45mL of the dye (Biosafe Coomassie blue) was placed on the gel. The instructor carried out the remaining method. The dye was allowed to sit on the get for an hour. Following, the gel was distained and photographed for personal home analysis using ImageJ …show more content…
Gel Electrophoresis of Samples. Gel was used to run the samples of each group. Given the sample assigned was F7, the circled samples had two distinct bands. Below the calculation was done to discover the molecular weight.
Sample: F7 Identified Protein: Bovine Serum Albumin (BSA)
Analysis: This is too heavy to be Immunoglobulin so analyzing one band was the better option. Looking at the second band it is relatively close to the weight of Bovine Serum Albumin so it was labeled as such.
Discussion:
1. The protein best matched with Bovine Serum Albumin (BSA).
2. The protein was just 3 kDa off from the literature value. Possible discrepancies could be from the run of the gel. Experimental errors were found as the standard latter only presented 8 instead of 10 band, and those values directly impacted the standard curve equation used to get the molecular weight.
3. The protein is associated with the following biological processes: cellular response to starvation, hemolysis by symbiont of host erythrocytes, maintenance of mitochondrion, negative regulation of apoptotic process, and transport. (cite
Introduction: The purpose of this experiment was to identify an unknown protein sample by SDS-Page. SDS-page is a shortened name for sodium dodecyl sulfate polyacrylamide gel that separated proteins based on the size of the molecules using negatively charged detergents (Huang 2016). The size of the molecule determined the distance traveled by the protein as larger proteins traversed shorter distance and visa versa. Due to size of the proteins being the main determination factor, it was essential to establish the same charge to mass ratio; specific reagents were used to meet this goal. β-Mercaptoethonol was used as a reducing agent to disrupt the tertiary structure and disconnect …show more content…
The samples were placed for five minutes in a 95 C heating block. After the instructor loaded the standard into the gel, members loaded their labeled protein samples into the appropriate electrophoresis wells. The cover of the apparatus was attached and the associated colored cables. The apparatus was slowly turned on to a voltage of 90 V and allowed to run. The standard loaded by the instructor was monitored as it was dyed and was used to measure the completion of the run. As the gel was ran, ImageJ software was explored on a sample picture of a ran gel to analyze the distance traveled. After about 40 minutes, the gel was removed and rinsed with deionized water for three minutes, and about 45mL of the dye (Biosafe Coomassie blue) was placed on the gel. The instructor carried out the remaining method. The dye was allowed to sit on the get for an hour. Following, the gel was distained and photographed for personal home analysis using ImageJ …show more content…
Gel Electrophoresis of Samples. Gel was used to run the samples of each group. Given the sample assigned was F7, the circled samples had two distinct bands. Below the calculation was done to discover the molecular weight.
Sample: F7 Identified Protein: Bovine Serum Albumin (BSA)
Analysis: This is too heavy to be Immunoglobulin so analyzing one band was the better option. Looking at the second band it is relatively close to the weight of Bovine Serum Albumin so it was labeled as such.
Discussion:
1. The protein best matched with Bovine Serum Albumin (BSA).
2. The protein was just 3 kDa off from the literature value. Possible discrepancies could be from the run of the gel. Experimental errors were found as the standard latter only presented 8 instead of 10 band, and those values directly impacted the standard curve equation used to get the molecular weight.
3. The protein is associated with the following biological processes: cellular response to starvation, hemolysis by symbiont of host erythrocytes, maintenance of mitochondrion, negative regulation of apoptotic process, and transport. (cite