Non-Abcam antibody was used: IRDye 800CW donkey anti-rabbit secondary ab Host species: Donkey Clonality: Polyclonal Conjugation: IRDye 800CW
LI-COR fluorescence channels
Negative control were the same amount of HEK293T cells, same procedure of harvesting, cleaning and lysis. Despite the fact others were transfected with GFP expression vectors this one was just treated with PEI reagent and NO DNA/vectors at all. However, these non transfected cells show a slight signal of endogenous PKA-C (at 38kDa) subunit if you pitch up the LI-COR fluo signal achieved with your abcam aPKA-Cgamma antibody
Specific: 69 kDa Non-specific: 48, 70, 50 kDa
SInce the probes where all 3 PKA C subunit isoforms, namely Alpha, Beta 1/2 and Gamma, there were no positive control. The specificity of this antibody and the lack of knowledge about other antibodies made it hardly possible to choose a positive control. However, we expressed all these isoforms as GFP-tagged proteins in HEK293T cells to detect it simultaneously with a anti-GFP antibody - which shows merged overlaps of GFP and PKA-Cgamma signals
HEK293T Cells were cultivated in a 6 well plate each transfected with different GFP-contructs namely PKA-alpha/beta1/Beta2/Gamma/Gamma mutant (All PKA-C subunits are roughly 41 kDa + the fusion GFP protein 27 kDa = 69.7 kDa including spacer) . They were seeded as 5E+5 cells per well for roughly 62 hours in DMEM + 10% FBS media. HArvesting was done with washing once with 1x PBS Buffer and scraping cells off the wells with buffer, centrifuged by 7000xg 5mins at RT and supplied each with 100uL 2x SDS buffer (+10% betaME).
The western blot was a semi dry one (1h 100mA). we had nitrocellulose membrane for blotting (GE HEalthcare). This was the blocking/dilution buffer for the antibodies supplied with 5% milk powder for blocking and 2% milk powder for over night (18h, rotating 10 rpm at 4 ¯C) incubation of your antibody: (50 mM Tris-HCl pH 7,5 150 mM NaCl 0,1 % (v/v) TWEEN 20).
Lanes are as following: M Chameleon Duo prestained, 1 GFP-PKA-Calpha, 2 GFP-PKA-Cbeta1, 3 GFP-PKA-Cbeta2, 4 GFP-PKA-Cgamma, 5 GFP-PKA-Cgamma mutant, 6 non transfecting cells. Left green blot is the CW800 fluo signal of your abcam PKA-Cgamma. On the right side is the same green signals merged with a red PKA-C pan antibody of Santa Cruz biotechnology swhich detects ALL three isoforms of PKA.
Abcam user community
Submitted Jan 29 2018
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