User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/23

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Immunoblot (95, 450, 250) Main project page
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Summary

Immunoblot

  • AKAP250 (+αRII) Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) (GOAT)
  • AKAP95 (+αRII) BD Biosciences (Heidelberg, Germany) (MOUSE)
  • AKAP450 (+αRII) BD Biosciences (Heidelberg, Germany) (MOUSE)
    • αRII BD Biosciences (Heidelberg, Germany) (MOUSE)


  • Primary antibodies were diluted 1:1000 and incubated for 1.75 h (9:30 - 11.15 AM) @ RT while shaking
    • Dilution occurred in blocking buffer (1% BSA in TBS-T)
  • Blots were washed 3x 10 min.
  • Secondary antibodies were diluted 1:10.000 and incubated for 1 h @ RT while shaking
  • Blots were washed, 1x 5 min. 1x 30 min. 1x 2 min. (due to meeting and time limitations on lumi)

Immunoblot part II

  • Blots used for AKAP250, AKAP95, AKAP450 and the RIIα blot of AKAP95 were blocked for 1 h in blocking buffer with Azide
    • 20% sodium azide in PBS was diluted 1:1000 in blocking buffer
  • And all were incubated with the same primary antibody ON @ 4 °C (diluted in blocking buffer + Azide)

Results

  • Immunoblot

Gel loading hTERT D9 + HEK293 see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10
Gel
cAMP prec.
X HEK293
(20 μL)
Marker
(8 μL)
C1
(20 μL)
C2
(20 μL)
C3
(20 μL)
S1
(20 μL)
S2
(20 μL)
S3
(20 μL)
X

Discussion

  • On the RII overlay of 19May2010 the higher bands were of a low concentration compared to the RII bands (~55 kDa). Perhaps too low to see on immunoblot?
    • Higher concentrations (>0.5 mg/mL total protein on cAMP beads) would not be necessary, because this would oversaturate the beads
  • Gravin (AKAP250) has been shown in HEK293 cells and are also not seen here, stating a problem with transfer or antibody
    • To make sure AKAPs >100 kDa are transferred to the membrane tank blotting should be used
    • Maybe new antibody? Gravin en Yotiao antibodies have just been produced and could be used (not tested)
  • Why RIIα could not be detected on only one of the blots is not known
  • Beads are working fine, because you can see RII subunits. Same for gel quality


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