03/01/2016
Western secondary antibody and imaging
FIRST
- Secondary antibody (same for each of the primaries I used)
- goat anti-rabbit IgG-HRP, santa cruz sc-2030, store at 4degC
- 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.
- 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
- 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.
- 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
- 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.
- 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
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.
Transfect Luc14s
g025, 34, 48, 55, triplicate
3ul lipo
let complexes form for 30 mins
didn't change media, kept the media with antibiotics in case the media change was messing things up
Cass picked 6 colonies from my MV14 ligation plate into 4mLs LB+amp
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