User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/05/31

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SDS-PAGE for AKAP79 immunoblot & RII overlay (HEK293) Main project page
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Summary

SDS-PAGE

Biorad Precision Plus Protein Dual Color marker
#1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
SampleMarkerhTERT RIPAhTERT PotterHEK293 RIPAMarkerhTERT RIPAhTERT Potter HEK293 RIPAxxxxxxx
#2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
SampleMarkerhTERT RIPAhTERT PotterHEK293 RIPAMarkerhTERT RIPAhTERT PotterHEK293 RIPAxxxxxxx

Splitting cells

  • Splitting cells into 12-wells?
  • D9 to passage 25

Immunoblot

  • Block membrane in 1% BSA TBST solution for >1 h @ RT
  • Incubate with primary antibody 2 h @ RT or ON @ 4 °C
    • 1:1000 dilution of antibody in blocking solution
  • Wash blot 10 min. with blocking solution
  • Incubate with secondary antibody 1 h @ RT
    • anti-rabbit, anti-mouse or anti-goat
    • 1:10.000 dilution in blocking solution
  • Wash 3x 10 min. with blocking solution

Preparing film

  • 1:1 ratio ECL solutions (2 mL per membrane)
  • Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
  • Place blot in between plastic
  • Take picture (depends on what is available)

Notes

  • Wells of gel #1 were not great in that they appeared not to be perfectly separated at the bottom
  • Gel #1 has the front of the loading buffer a bit blurry around 40 ~ 75 kDa
  • Gel #1 took forever to run (3h and stil not completely done), and it was not the only one in the lab, so diffulties in SDS-PAGE not completely gone. Gel #2 cancelled for today

Results

  • WHAT?

Discussion

  • DONE Ow..


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