Western blot abreview for Anti-PKA gamma (catalytic subunit) antibody [EP2647Y]

Average
Abreviews
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293T cell line)
Gel Running Conditions
Reduced Denaturing (12 % gel)
Loading amount
2e+006 cells
Specification
HEK293T cell line
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Other product details

Incubation time
18 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: TBS + Tween20
Dilution
1/50000

Secondary antibody

Dilution
1/15000
Name
Non-Abcam antibody was used: IRDye­ 800CW donkey anti-rabbit secondary ab
Host species: Donkey
Clonality: Polyclonal
Conjugation: IRDye­ 800CW

Detection

Detection method
LI-COR fluorescence channels
Negative control
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
Exposure
35 second(s)
Bands
Specific: 69 kDa Non-specific: 48, 70, 50 kDa
Positive control
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

Additional data

Additional Notes
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.

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Submitted Jan 29 2018

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