4 Polyacrylamide Gel Electrophoresis of Serum Proteins and Identification of Albumin Proteins by Western Blotting PDF

Preview

Summary

4 Polyacrylamide Gel Electrophoresis of Serum Proteins and Identification of Albumin Proteins by Western Blotting...

Description

Polyacrylamide Gel Electrophoresis of Serum Proteins and Identification of Albumin Proteins by Western Blotting

I.

Pre-Laboratory. a) What scientific concept(s) will be learned in this lab activity?  The scientific concepts that will be learned in this lab are the preparation of polyacrylamide gel electrophoresis and the identification of albumin proteins. b) What laboratory techniques will be learned in this activity?  The techniques that are covered in this lab are to prepare gel electrophoresis and insert it into the electrode assembly to create the mini protean assembly. Other techniques such as loading the gel and running it after dying to show the lanes. c) What is the objective (or goal) for the lab?  The goal for this lab is for students to learn how to interpret data on a polyacrylamide and how to detect serum albumin protein on the stained gel. Also the goal for this lab is to separate and compare protein from mammalian species.

II.

Materials and Methods.s Dr. Pattison’s BCHS 3201 protocol, pages 67-84. Western Blotting was not performed in this experiment and therefore, only one gel was required for the lab. The native gel was used instead of SDS gel. When adding a runner buffer to the mini tank, it was measured halfway during lab without accuracy unlike the protocol that specifies 200 ml.

III.

Results. Picture of the gel is on the next page.

Picture 1- Stained Serum Albumin Protein: gel electrophoresis is shown above with mammalian species sera and markers. Each lane specifies a mammalian serum with the outer lanes left empty. IV. Discussion/Post-Lab Questions. In picture 1, the gel electrophoresis with mammalian serum and markers are shown. By looking at the picture, it can be seen that these mammals are closely related and that can be seen from the bands and where they are concentrated on the gel. There are multiple bands in this picture which lead to the conclusion that the proteins migrated down the gel. The heavier proteins are concentrated towards the top near the marks of the comb whereas the lighter proteins are more towards the bottom because it is easier for them to travel along the gel. The kaleidoscope lane shows a variety of proteins by molecular weight that could be depicted from the proteins migration across the gel. The human and the monkey sera have very similar bands and that is due to the close relationship between the species. For the BSA lane, there are almost no bands shown in that lane. Serum Albumin protein is then detected across the band. It is a small molecule with a lightly negative charge and that is why it accumulates on the gel in the first bands shown which is also pointed by the arrow on the picture on the Results part.  Post Lab Questions: 1. Label your gel picture. Each lane should be labeled. Circle the bands that most likely correspond to serum albumin proteins.  This picture is shown in the Results part.

2. Describe the results of our Coomasie-stained gel. What is in each lane? Do you have multiple bands or single bands? Why?  Looking at the picture, the outer lanes were left empty. Starting from the second lane on the left: kaleidoscope, human, this lane left empty, monkey, pig, horse, bovine, and then BSA sera are loaded in the lanes. It can be seen that each species is very similar in types of proteins that are shown. Not only as the type but the concentration and molecular weight are displayed though what bands are available. Multiple bands are present as well as single bands. The bands are indication or presence of proteins in every species serum. All the lanes have multiple bands except for the BSA one which has only single bands. There are circled stained down the gel which indicates that there are large proteins that migrated down the gel. The reason for showing multiple and single bands is the denatured structure of the proteins present in all the sera. 3. What purpose does the Kaleidoscope marker serve in your Coomassie-stained gel? If you were doing a Western blot, how would the Kaleidoscope marker be useful?  The purpose of this marker is to make sure the gel is running properly in this lab. It serves to help visualize the different molecular weights that migrated across the gel. As for the Western Blot, this marker is used to act as an indicator of whether or not the transfer of protein to the nitrocellulose is completed. Also, this marker is useful to approximate the molecular weight of the proteins on the gel. 4. Which lanes had bands in the sample Western blot your TA showed from this experiment? Why?  The sample Western blot only showed bands from the monkey serum and human serum. These two lanes showed bands because as expected, the primary antibody attached to the protein of interest, the second antibody attached to the primary antibody, and the colorimetric enzyme substrate reacted with the second antibody, therefore making it noticeable and developing the bands that are shown. 5. You accidentally use an SDS-PAGE system to do this experiment. What impact would this have on your Western blot results?  If SDS-page gel was to accidentally be used in this experiment, the proteins will be maintained in a denatured state and not in their native-folded conformation. As a result, the antibodies cannot bind in the Western blot which will decrease the resolution. So polyacrylamide gel is better to use than the agarose gel. 6. If you wanted to determine the molecular weight of albumin, how would you change the experimental protocol?  Further isolation need to be considered to determine the molecular weight. A possibility could be to use SDS Polyacrylamide Gel instead of a Native Gel. SDS Polyacrylamide Gel causes the protein to denature and have corresponding charge to mass ratios. This might help to determine molecular masses more easily....