Identification of serum glycoprotein by polyacrylamide gel electrophoresis (PAGE) and western blotting

Identification of serum glycoprotein by polyacrylamide gel electrophoresis (PAGE) and western blotting.

Identification of serum glycoprotein by polyacrylamide gel electrophoresis (PAGE) and western blotting

Prelab

Protein electrophoresis is used to determine the protein present in a fluid or extract. Only a small volume of the sample is needed. Native gel electrophoresis or PAGE usually analyses proteins that are in their folded state. It does not use denaturing gels. Separation of proteins is due to their charge to mass ratio, shape and size. Coomasie blue stain is used in staining to visualize the separated proteins on the gel. It is however, not very sensitive and silver staining is more sensitive but is toxic. The size of pores on the gel is very important usually the ore size is dictated by the ratio of bisacrylamide to polyacrilamide.

Western bolt is used to detect specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis (PAGE) to separate native proteins that are in the original conformation. This technique helps to ensure that the proteins expected to have been separated were separated using gel electrophoresis. Proteins separated from PAGE are transferred to a membrane and then stained with antibodies specific to the target protein (Vallejo‐Illarramendi, 1148, 1149, 1150).

The objective of this study therefore, is to separate proteins based on PAGE and further detect the proteins of a western blot.

Materials and methods

Each step in this practical activity was performed according to Dr. Pattinson’s protocol

 

Results

6
5
4
3
2
1
Serum proteins separation.
No protein

separation

Marker

The above diagram is an image of the separation of proteins on the PAGE performed.

Discussion

The proteins as seen in the results were well separated on the PAGE the large proteins moved slowly as they were restricted by the size of the pores on the gel while the smaller proteins moved faster down the gel as they were less restricted by the gel.

In the lanes 2, 3, 4 and 5 of the gel is the separated protein except for the lane number 1 where there is a ladder that helps to assess the size of the bands with multiple bands due to the fact that serum contains albumin and globulin. Lane 6 did not have proteins. The only lanes that would have shown proteins on western blot are lane 2, 3, 4 and 5.

The molecular weight of albumin is 66.5kDa much larger than globulin which can range from 90-1200 kDa. Albumin is the most abundant protein as compared to globulin and this two albumins and globulin are the proteins found in serum. It appears on the gel in abundance. Coomasie blue staining is not the best staining method in identification of proteins on a gel because it has low sensitivity silver staining is more effective but the silver stain is toxic and hence the reason why coomasie blue is preferred.

Therefore, proteins in a fluid can be separated using electrophoresis and further detected using western blot.


 

Reference

Vallejo‐Illarramendi, Ainara, Denise K. Marciano, and Louis F. Reichardt. “A novel method that improves sensitivity of protein detection in PAGE and Western blot.” Electrophoresis 34.8 (2013): 1148-1150.

Identification of serum glycoprotein by polyacrylamide gel electrophoresis (PAGE) and western blotting

Prelab

Protein electrophoresis is used to determine the protein present in a fluid or extract. Only a small volume of the sample is needed. Native gel electrophoresis or PAGE usually analyses proteins that are in their folded state. It does not use denaturing gels. Separation of proteins is due to their charge to mass ratio, shape and size. Coomasie blue stain is used in staining to visualize the separated proteins on the gel. It is however, not very sensitive and silver staining is more sensitive but is toxic. The size of pores on the gel is very important usually the ore size is dictated by the ratio of bisacrylamide to polyacrilamide.

Western bolt is used to detect specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis (PAGE) to separate native proteins that are in the original conformation. This technique helps to ensure that the proteins expected to have been separated were separated using gel electrophoresis. Proteins separated from PAGE are transferred to a membrane and then stained with antibodies specific to the target protein (Vallejo‐Illarramendi, 1148, 1149, 1150).

The objective of this study therefore, is to separate proteins based on PAGE and further detect the proteins of a western blot.

Materials and methods

Each step in this practical activity was performed according to Dr. Pattinson’s protocol

 

Results

6
5
4
3
2
1
Serum proteins separation.
No protein

separation

Marker

The above diagram is an image of the separation of proteins on the PAGE performed.

Discussion

The proteins as seen in the results were well separated on the PAGE the large proteins moved slowly as they were restricted by the size of the pores on the gel while the smaller proteins moved faster down the gel as they were less restricted by the gel.

In the lanes 2, 3, 4 and 5 of the gel is the separated protein except for the lane number 1 where there is a ladder that helps to assess the size of the bands with multiple bands due to the fact that serum contains albumin and globulin. Lane 6 did not have proteins. The only lanes that would have shown proteins on western blot are lane 2, 3, 4 and 5.

The molecular weight of albumin is 66.5kDa much larger than globulin which can range from 90-1200 kDa. Albumin is the most abundant protein as compared to globulin and this two albumins and globulin are the proteins found in serum. It appears on the gel in abundance. Coomasie blue staining is not the best staining method in identification of proteins on a gel because it has low sensitivity silver staining is more effective but the silver stain is toxic and hence the reason why coomasie blue is preferred.

Therefore, proteins in a fluid can be separated using electrophoresis and further detected using western blot.


 

Reference

Vallejo‐Illarramendi, Ainara, Denise K. Marciano, and Louis F. Reichardt. “A novel method that improves sensitivity of protein detection in PAGE and Western blot.” Electrophoresis 34.8 (2013): 1148-1150.

 

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Identification of serum glycoprotein by polyacrylamide gel electrophoresis (PAGE) and western blotting