Anti-Caspr antibody (ab34151)

All lanes : Anti-Caspr antibody (ab34151) at 1/250 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CNTNAP1 (Caspr) knockout HAP1 whole cell lysate
Lane 3 : MEF whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 156 kDa
Observed band size: 180 kDa (why is the actual band size different from the predicted?)

Lanes 1 - 4: Merged signal (red and green). Green - ab34151 observed at 180 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab34151 was shown to recognize Caspr in wild-type HAP1 cells as signal was lost at the expected MW in CNTNAP1 (Caspr) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CNTNAP1 (Caspr) knockout samples were subjected to SDS-PAGE. ab34151 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/250 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

Anti-Caspr antibody (ab34151) at 1/250 dilution + Brain (Rat) Whole Cell 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: 156 kDa
Observed band size: 180 kDa (why is the actual band size different from the predicted?)
Additional bands at: 58 kDa. We are unsure as to the identity of these extra bands.



Caspr contains a number of potential glycosylation sites so it is thought that this is the reason it runs at 180kDa.

ICC/IF image of ab34151 stained SHSY5Y cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab34151, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

IHC-FoFR image of ab34151 stained sections of mouse brain (30 um). The tissues were from perfused fixed animals perfused with 4% PFA and postfixed 2h in the same fixative. They were cryoprotected in 30% sucrose and cut using a cryostat.

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Caspr was immunoprecipitated using 0.5mg Rat Brain whole tissue lysate, 5µg of Rabbit polyclonal to Caspr 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, Rat 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 ab34151.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 180kDa: Caspr.

ab34151 staining Caspr in murine brain tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with paraformaldehyde. Samples were blocked with 20% serum for 1 hour at 20°C followed by incubation with the primary antibody at a 1/300 dilution for 12 hours at 20°C. An HRP-conjugated goat anti-rabbit polyclonal was used as the secondary antibody at a 1/200 dilution.

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