Western Blot Method - Animated Video — Transcript

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1. 0:00Western blotting is an important technique used in cell and molecular biology
2. 0:04for protein separation and detection
3. 0:07it enables the separation and identification of a specific protein of interest
4. 0:11from a complex mixture of proteins
5. 0:13for example a cell lysate
6. 0:15A typical Western blot technique includes the following steps
7. 0:19separation by size, transfer of protein to a solid support
8. 0:23and detecting target protein using antibodies
9. 0:27to separate the macromolecules in a sample
10. 0:29SDS-PAGE technique can be used
11. 0:32The first step in the SDS-Page procedure is sample preparation
12. 0:37So that, a loading buffer containing SDS, betamercaptoethanol
13. 0:42bromophenol blue and glycerol is added to the protein samples
14. 0:48the loading buffer is used in order to give all proteins present a uniform negative charge
15. 0:54since proteins can be positively, negatively, or neutrally charged
16. 1:01Once the loading buffer is added
17. 1:03the samples are then heated to 95 degrees Celsius
18. 1:09Proteins are large biomolecules, consisting of one or more long chains of amino acid residues
19. 1:15they are formed by linking amino acids with peptide bonds
20. 1:19and fold into specific spatial conformations driven by a number of interactions
21. 1:23such as Hydrogen bonds, Hydrophobic interaction, Disulfide bonds, Ionic bonds.
22. 1:30SDS is an anionic surfactant that contain a polar head group with a net negative charge
23. 1:35at the end of a long hydrophobic carbon chain
24. 1:38SDS denatures the native proteins by disturbing the non-covalent forces
25. 1:43include hydrogen-bonding, hydrophobic and ionic interactions
26. 1:48While the reducing agent betamercaptoethanol is used to cleave the disulfide bonds
27. 1:55Also, SDS binds fairly uniformly to the protein
28. 1:58and the intrinsic charges of this protein become negligible
29. 2:01when compared to the negative charges contributed by SDS
30. 2:05this treatment brings the folded proteins down to linear molecules, with net negative charge
31. 2:12therefore, these proteins can be separated in a polyacrylamide gel based on their molecular weight
32. 2:20The gel is produced by polymerisation between two glass plates anchored vertically in a cassette
33. 2:28The gel cassette is placed vertically between two electrodes
34. 2:31positive electrode located at the bottom of the gel
35. 2:34whereas negative electrode is positioned at the top of the gel
36. 2:40Next, the gel is inserted into a chamber
37. 2:44then a running buffer is poured to allow the conduction of current through the gel
38. 2:48SDS is also present in the gel and in the running buffer, to make sure that
39. 2:52once the proteins are denatured, they stay that way throughout the run
40. 2:56During separation, a molecular-weight size marker is usually loaded onto the gel
41. 3:01This consists of proteins of known sizes
42. 3:04and thereby allows the estimation of the sizes of the proteins in the actual samples
43. 3:08which migrate in parallel in different tracks of the gel
44. 3:15Each sample is added into its own well in the gel
45. 3:18as we have seen previously, bromophenol blue and glycerol are present in the samples
46. 3:24bromophenol blue is a dye, that is useful for visualizing the sample in the well
47. 3:28while, Glycerol increases the density of a sample and it is used to fall the sample into the well
48. 3:39after the sample application procedure, an electric field is applied across the gel
49. 3:48causing the negatively charged proteins to migrate across the gel
50. 3:52away from the negative electrode, and towards the positive electrode
51. 3:59Also, Small proteins migrate relatively easily through the mesh of the gel
52. 4:04while larger proteins are more likely to be retained and thereby migrate more slowly
53. 4:10Due to the relatively small molecule size of bromophenol blue, it migrates faster than proteins
54. 4:16and by optical control of the migrating-colored band
55. 4:19the electrophoresis can be stopped before the samples have completely migrated through the gel and leave it
56. 4:26Once the proteins are separated, the gel cassette is removed from the electrophoresis tank
57. 4:32Next, the glass plates are removed
58. 4:35and, the top portion containing the wells is cut off from the gel
59. 4:39In the next step, the separated proteins will be transferred
60. 4:42from inside the gel to an appropriate membrane
61. 4:45When performing a wet transfer, the gel is first equilibrated in transfer buffer
62. 4:51so that, it is placed in a tray with the blotting buffer, on a rocking platform
63. 4:59To make a blotting sandwich, a gel holder cassette is immersed in the transfer buffer
64. 5:03with the black side down and the white side up, and out of the buffer
65. 5:07Next, a fiber foam pad is immersed in the transfer buffer
66. 5:12Then, it is laid on the black side of the gel holder cassette
67. 5:16After that, a piece of a filter paper is soaked in the transfer buffer
68. 5:20Then, it is placed on top of the fiber pad
69. 5:23once the gel has been equilibrated in the transfer buffer
70. 5:27it is carefully placed on the blotting paper
71. 5:32For the transfer, a nitrocellulose membrane or a polyvinylidene difluoride membrane can be used
72. 5:37the membrane is soaked in the transfer buffer
73. 5:40then, it is placed squarely on the gel
74. 5:43next, a second sheet of blotting paper is wetted with the transfer buffer
75. 5:47Then, it is placed on top of the membrane
76. 5:51Finally, a second fiber pad is immersed in the transfer buffer
77. 5:54Then, it is laid on top of the blotting paper
78. 5:58Next, the gel holder cassette is closed and locked with a clamp
79. 6:03The most commonly used method for transferring the proteins is called electroblotting
80. 6:08During this transfer, an electrode assembly with positive electrode and negative electrode is used
81. 6:15The gel holder cassette is inserted into the module
82. 6:18ensuring that the cassette is properly positioned from negative to positive
83. 6:22The inner module is placed into the electrophoresis chamber
84. 6:27then, the chamber is filled with the blotting buffer
85. 6:33Next, a lid is placed on the electrophoresis tank
86. 6:36and electrical leads are connected to the power supply to run the blot
87. 6:43Electroblotting uses an electric current to pull the negatively charged proteins from the gel
88. 6:47towards the positively charged electrode, and into the membrane
89. 6:52The proteins move from within the gel onto the membrane
90. 6:55while maintaining the organization they had within the gel
91. 6:58when the run is complete, the electrophoresis chamber is disassembled
92. 7:02and the inner module is removed
93. 7:05Then, the gel holder cassette is removed and opened
94. 7:09Next, starting with the fiber pad, each layer is removed until reaching the membrane
95. 7:17After the electrotransfer of the proteins on the membrane
96. 7:20the next step is the detection of our protein of interest using antibodies
97. 7:26first of all, the membrane is soaked in a blocking solution
98. 7:29which contains bovine serum albumin
99. 7:33this step must be taken to prevent the interactions between the membrane
100. 7:37and the antibody used for detection of the target protein
101. 7:40once the blocking solution has been added, the membrane is incubated on a rocking platform.
102. 7:46The BSA protein attaches to the membrane in all places where the target proteins have not attached
103. 7:52Thus, when the antibody is added, it cannot bind to the membrane
104. 7:55and therefore the only available binding site is the specific target protein
105. 8:00After the incubation is complete, the blocking solution is removed
106. 8:05then a solution containing the primary antibody is poured into the tray
107. 8:13next the membrane is incubated under gentle agitation
108. 8:20The primary antibody binds to the specific target protein
109. 8:31Following incubation, the primary antibody solution is removed
110. 8:36then a wash buffer is poured
111. 8:42next the membrane is incubated on the rocking platform
112. 8:47the membrane is washed several times in wash buffer to remove unbound primary antibody
113. 8:52and thereby minimize background
114. 8:58after washing the membrane, the wash buffer is removed
115. 9:03then, a solution containing the secondary antibody is added into the tank
116. 9:14next the membrane is incubated under gentle agitation
117. 9:18The secondary antibody recognises and binds to a specific portion of the primary antibody
118. 9:28Following incubation, the secondary antibody solution is removed
119. 9:32then, the wash buffer is poured into the tank
120. 9:40next the membrane is incubated on the rocking platform
121. 9:46the membrane is washed several times in wash buffer to remove unbound secondary antibodies
122. 9:51It is crucial to thoroughly wash the membrane at this step
123. 9:56After rinsing the membrane to remove unbound secondary antibody
124. 10:02the membrane is incubated in a solution containing the substrate.
125. 10:08Chemiluminescent western blot detection is the most frequently used method
126. 10:12to detect proteins on Western blots
127. 10:16To allow detection of the target protein
128. 10:18the secondary antibody is commonly linked to a reporter enzyme such as horseradish peroxidase
129. 10:25The most popular Western blot substrates are luminol-based
130. 10:31in the presence of a hydrogen peroxide, Horseradish peroxidase enzyme catalyses the oxidation of luminol
131. 10:37to excited state product called 3-aminophthalate
132. 10:41This product decays to a lower energy state by releasing photons of light at 425 nm
133. 10:50the production of luminescence is proportional to the amount of horseradish peroxidase-conjugated secondary antibody
134. 10:56and therefore, indirectly measures the presence of the target protein
135. 11:02After incubation, the membrane is placed in a clear plastic wrap to prevent drying
136. 11:12The membrane is analysed by densitometry
137. 11:15where the resulting light signal is detected by CCD camera which captures a digital image of the western blot
138. 11:25according to the obtained bands, we can determine the approximate size of our protein of interest
139. 11:31as well as its presence or absence in each sample