Western Blotting: Difference between revisions

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==SOP==

*~~Irritant~~

* [[SOP_-_Irritants|SOP - Irritants]]

*Electrophoresis

* [[SOP - Electrophoresis]]

==Materials==

*Protein Markers, we use SeaBlue Plus2 from Invitrogen (cat #LC5925)

*Nitrocellulose, we use BioRad 0.45 um membrane from a roll (cat #1620115) and cut to the size of a gel

*Filter Paper, we use BioRad extra thick paper (cat 1703966)

*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)

*Transfer Apparatus, either Bio-Rad or Invitrogen

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#Turn on heat block to 85 degrees

#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer

## Use a prepared ~~4-12% tris~~ gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.

## Use a prepared gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water. Pick a percent gel based on your expected protein size.

##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back

##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back. For a video on how to use these gel tanks see https://youtu.be/aedIjTtNEus

##Boil sample at 85 degrees for ~3 min

##Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.

@@ Line 1: / Line 1: @@
 ==SOP==
-*Irritant
+* [[SOP_-_Irritants|SOP - Irritants]]
-*Electrophoresis
+* [[SOP - Electrophoresis]]
 ==Materials==
+*Protein Markers, we use SeaBlue Plus2 from Invitrogen (cat #LC5925)
+*Nitrocellulose, we use BioRad 0.45 um membrane from a roll (cat #1620115) and cut to the size of a gel
+*Filter Paper, we use BioRad extra thick paper (cat 1703966)
 *Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
 *Transfer Apparatus, either Bio-Rad or Invitrogen
@@ Line 10: / Line 13: @@
 #Turn on heat block to 85 degrees
 #Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
-## Use a prepared 4-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.
+## Use a prepared gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.  Pick a percent gel based on your expected protein size.
-##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
+##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back.  For a video on how to use these gel tanks see https://youtu.be/aedIjTtNEus
 ##Boil sample at 85 degrees for ~3 min
 ##Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.