ab31704 staining DCAMKL1 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab31704 at 5 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: DCAMKL1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: NIH3T3 whole cell lysate (20 µg)
Lane 4: Human brain whole tissue lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab31704 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab31704 was shown to specifically react with DCAMKL1 in wild-type HAP1 cells along with additional cross reactive bands. No bands were observed when DCAMKL1 knockout cells were examined. Wild-type and DCAMKL1 knockout samples were subjected to SDS-PAGE. ab31704 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 µg/ml and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

DCAMKL1 was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 5µg of Rabbit polyclonal to DCAMKL1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain whole tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31704.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 82kDa: DCAMKL1; non specific - 52 and 27kDa: We are unsure as to the identity of this extra band.

Anti-DCAMKL1 antibody (ab31704) at 1 µg/ml + Mouse Brain Whole Tissue Lysate at 20 µg

Secondary
IR Dye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Performed under reducing conditions.

Predicted band size: 47, 82 kDa
Observed band size: 47,82 kDa why is the actual band size different from the predicted?



The 82 kDa and 47 kDa bands correspond to the AL and BL isoforms respectively. This antibody should not detect the AS and BS isoforms of DCAMLK1.

ICC/IF image of ab31704 stained PC12 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab31704, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue).

Anti-DCAMKL1 antibody (ab31704) at 1 µg/ml + Brain (Rat) Tissue Lysate - normal tissue at 10 µg

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 47, 82 kDa
Observed band size: 47,82 kDa why is the actual band size different from the predicted?

Anti-DCAMKL1 antibody (ab31704) at 1 µg/ml + Human brain tissue lysate - total protein (ab29466) at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 47, 82 kDa
Observed band size: 47,82 kDa why is the actual band size different from the predicted?
Additional bands at: 30 kDa, 52 kDa (possible post-translational modification), 54 kDa (possible post-translational modification). We are unsure as to the identity of these extra bands.


Exposure time: 5 minutes


The 82 kDa and 47 kDa bands correspond to the AL and BL isoforms respectively. DCAMLK1 has a number of potential phosphorylation sites which may explain the higher migrating bands at 52 and 54 kDa.