Haynes Lab:Notebook/CRISPR Editing/2016/03/01: Difference between revisions
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'''Western secondary antibody and imaging'''<br> | '''Western secondary antibody and imaging'''<br> | ||
FIRST | FIRST | ||
- | - Secondary antibody (same for each of the primaries I used) | ||
:goat anti-rabbit IgG-HRP, santa cruz sc-2030, store at 4degC | :goat anti-rabbit IgG-HRP, santa cruz sc-2030, store at 4degC | ||
<br> | <br> | ||
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#Label THREE petri dishes with the antibodies you stained with. Do not place the blots into the same dish. | #Label THREE petri dishes with the antibodies you stained with. Do not place the blots into the same dish. | ||
#Remove each blot from its pouch and place it in the right petri dish | #Remove each blot from its pouch and place it in the right petri dish | ||
#Fill each dish with PBST until it is covered, and so that the buffer will not splash out when it is moved around. | #Fill each dish with PBST until it is covered, and so that the buffer will not splash out when it is moved around. (9mLs) | ||
#Wash at room temp for 10 min by placing the dishes onto the orbital platform shaker at medium-high speed. You want good action in the buffer to aggressively wash of non-specific binding, but not to hard the buffer splashes out and the corners of the blot get damaged. | #Wash at room temp for 10 min by placing the dishes onto the orbital platform shaker at medium-high speed. You want good action in the buffer to aggressively wash of non-specific binding, but not to hard the buffer splashes out and the corners of the blot get damaged. | ||
#Carefully pour off the PBST, leave the blots in the dish. Add new PBST, repeat the whole wash process three more times (4 washes total). | #Carefully pour off the PBST, leave the blots in the dish. Add new PBST, repeat the whole wash process three more times (4 washes total). | ||
<br><br> | <br><br> | ||
'''SECONDARY STAINING''' | '''SECONDARY STAINING''' | ||
#Same procedure as the primary, except | #Same procedure as the primary, except at a higher dilution of the secondary ab (see Benchling: 12/22/15 Western blot optimization). Incubate on the rotator at ROOM TEMP for exactly one hour. | ||
#Do the four washes the same way again. After the final wash, leave the blots in the PBST until ready to image<br><br> | :antibody is at a concentration of 200ug/0.5mL = 400ug/mL = 400ng/ul | ||
:instructins for the HRP staining protocol say concentration range for secondary should be 2-10ng/mL | |||
:staining in 2mL of buffer so need 4-20ng | |||
:dilute antibody in buffer 1:100, new concentration is 4ng/ul | |||
:decide on 10ng/2mL, need 2.5ul of diluted antibody per 2mL pouch | |||
#Do the four washes the same way again but for 15 mins each. After the final wash, leave the blots in the PBST until ready to image<br><br> | |||
'''STAINING HRP'''<br> | '''STAINING HRP'''<br> | ||
pipette 250ul of each into a bubble on a flat plastic lid | pipette 250ul of each into a bubble on a flat plastic lid |
Revision as of 11:25, 1 March 2016
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03/01/2016Western secondary antibody and imaging
STAINING HRP
set the membrane protein side down so it soaks in the bubble. drag the membrane until the bottom edge is just touching the bubble, then slowly lay the membrane across.
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