Haynes Lab:Notebook/CRISPR Editing/2016/02/24: Difference between revisions
Rene M Davis (talk | contribs) |
Rene M Davis (talk | contribs) |
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- Record the proper secondary for each primary...Make sure the secondary is conjugated to HRP. For instance "goat anti-rabbit HRP" will bind to any rabbit polyclonal primary. | - Record the proper secondary for each primary...Make sure the secondary is conjugated to HRP. For instance "goat anti-rabbit HRP" will bind to any rabbit polyclonal primary. | ||
:goat anti-rabbit IgG-HRP, santa cruz sc-2030 | :goat anti-rabbit IgG-HRP, santa cruz sc-2030, store at 4degC | ||
<br> | <br> | ||
#Make PBST (1x PBS-tween) using the 10x PBS from the shelf and the tween I gave you yesterday. The bottle for the PBST is on my shelf. Reuse this bottle. Use DI water. Do not use the expensive water. | #Make PBST (1x PBS-tween) using the 10x PBS from the shelf and the tween I gave you yesterday. The bottle for the PBST is on my shelf. Reuse this bottle. Use DI water. Do not use the expensive water. | ||
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#Same procedure as the primary, except use a 1:1000 dilution of the secondary ab (see Benchling: 12/22/15 Western blot optimization). Incubate on the rotator at ROOM TEMP for exactly one hour. | #Same procedure as the primary, except use a 1:1000 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 I get there and I will show you how to detect the HRP.<br><br> | #Do the four washes the same way again. After the final wash, leave the blots in the PBST until I get there and I will show you how to detect the HRP.<br><br> | ||
'''STAINING HRP'''<br> | |||
pipette 250ul of each into a bubble on a flat plastic lid | |||
:Thermo supersignal west 1856192 | |||
:Thermo supersignal west 1856191<br> | |||
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. <br> | |||
let sit for 2-10 minutes before imaging. | |||
'''IMAGING''' | '''IMAGING''' | ||
preset: femto west w overlay<br> | preset: femto west w overlay<br> | ||
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image. was difficult to get a good pic because our background was really high. can try to fix by setting the exposure area to the highest signal. | image. was difficult to get a good pic because our background was really high. can try to fix by setting the exposure area to the highest signal. | ||
<br><br> | <br><br> | ||
conclusion: signal was really high, likely because the blocking buffer was less stringent than when KAH did hers with milk. can compare this image to the ponceau stain, see if the ratio of the stained bands is higher. should redo with much less secondary antibody, maybe dilute 1:3000 or more. | '''conclusion:''' background signal was really high, likely because the blocking buffer was less stringent than when KAH did hers with milk. can compare this image to the ponceau stain, see if the ratio of the stained bands is higher. should redo with much less secondary antibody, maybe dilute 1:3000 or more. | ||
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__NOTOC__ | __NOTOC__ |
Revision as of 12:19, 27 February 2016
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02/24/2016Western 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. |