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

 .todo { color: red } .done { color: green} {| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Immunoblot (95, 450, 250)
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

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

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


 * }