Haynes Lab:Notebook/CRISPR Editing/2016/02/24

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02/24/2016

Western secondary antibody and imaging
FIRST - 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, store at 4degC


  1. 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.
  2. Label THREE petri dishes with the antibodies you stained with. Do not place the blots into the same dish.
  3. Remove each blot from its pouch and place it in the right petri dish
  4. Fill each dish with PBST until it is covered, and so that the buffer will not splash out when it is moved around.
  5. 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.
  6. 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).



SECONDARY STAINING

  1. 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.
  2. 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.

STAINING HRP
pipette 250ul of each into a bubble on a flat plastic lid

Thermo supersignal west 1856192
Thermo supersignal west 1856191

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.
let sit for 2-10 minutes before imaging.

IMAGING preset: femto west w overlay
take the UV box out, put the black background thing in
get a square dish to image the membrane in. remove one membrane from buffer, dab the edge with a kimwipe to remove extra liquid, place protein side up in dish
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.

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.



Cut 2ug MV13 KAH60 plasmid with 0.5ul SgrAI in Cutsmart for 25 minutes. Heat inactivated, ran 0.5ul on gel
File:Screenshot 2016-03-12 20.18.45