User:Karmella Haynes/Notebook/Polycomb project/2010/02/18

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02/18/10

 * &#x2713; Western: Polycomb-ATF lines (new 10-well gel)

Western blot

> SDS-PAGE --> Use new 10-well tris-glycine (4-20%) pre-cast gel --> Samples (25 μL total vol.): --> Make 100 mM DTT in 2x loading buffer (200 μL total; > enough for 6 μL/ gel lane) --> Add 12.5 μL DTT + buffer to each protein sample --> Incubate at 100°C/ 5 min. --> Run gel @ 120 V until dye front reaches the bottom
 * Pre-stained ladder: 20 μL
 * 126-1 - 132-8, FTRx: 12.5 μL protein
 * 133-1: 6.25 μL protein (+6.25 dH2O)

> Electroblot --> Run @ 100 V for 1 hr., 30 min.

> Immunostaining #1 --> Primary: mouse anti-myc tag, 1:5000, 10 mL (4°C/ overnight) --> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.) --> RESULT: Blank. Strip filter in 1x PBST overnight and re-probe w/ anti-DsRed

> Immunostaining #2 (2/22/10) --> Primary: rabbit anti-DsRed, 1:4000, 10 mL (4°C/ overnight) --> Secondary: donkey anti-rabbit IgG-HRP, 1:5,000 (R.T./ 1 hr.) (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)

--> Looks great. 131-9 not expressed very well --> Strip o/n and re-probe w/ 1:1000 anti-myc

> Immunostaining #3 (2/24/10) --> Primary: mouse anti-myc tag, 1:1000, 10 mL (4°C/ overnight) --> Secondary: donkey anti-mouse-HRP, 1:5,000 (R.T./ 1 hr.)

--> Non-specific signal


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