Anti-Caspr antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Caspr antibody (AB34151)

ab34151 staining Caspr in primary rat neurons/glia, DIV14 (prepared from E18 rat hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. SDHEP) cells. The cells were fixed with 100% methanol (5 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 ab34151 at 1µg/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).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Caspr antibody (AB34151)

ab34151 stained in SKNSH cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab34151 at 1μg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 μM for 1hour at room temperature.

• IP

Unknown

Immunoprecipitation - Anti-Caspr antibody (AB34151)

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.

All lanes:

Immunoprecipitation - Anti-Caspr antibody (ab34151)

Predicted band size: 156 kDa

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• WB

Supplier Data

Western blot - Anti-Caspr antibody (AB34151)

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.

All lanes:

Western blot - Anti-Caspr antibody (ab34151) at 1/250 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

CNTNAP1 (Caspr) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

MEF whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Predicted band size: 156 kDa

Observed band size: 180 kDa

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• WB

Project2301****

Western blot - Anti-Caspr antibody (AB34151)

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

All lanes:

Western blot - Anti-Caspr antibody (ab34151) at 1/250 dilution

All lanes:

Brain (Rat) Whole Cell Lysate - normal tissue at 10 µg

Secondary

All lanes:

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

Predicted band size: 156 kDa

Observed band size: 180 kDa,58 kDa

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• WB

CiteAb

Western blot - Anti-Caspr antibody (AB34151)

Caspr western blot using anti-Caspr antibody ab34151. Publication image and figure legend from Lee, J. Y., Kim, M. J., et al., 2017, Sci Rep, PubMed 28827698.

ab34151 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab34151 please see the product overview.

Reduced interaction between PrPC and Caspr in ngr1-/- mice. (A–C) Western blot of LSSC lysates from ngr1+/+ and ngr1-/- mice showed no difference in Reelin expression. (A) Representative immunoblots for Reelin and Actin loading control. (B) Densitometric quantification of full-length Reelin (FL Reelin) and (C) 140 kDa degradation product of Reelin and Actin respectively. (D) Immunoprecipitation of Caspr showed reduced interaction with PrPC in LSSC from ngr1-/- mice. Western blot for PrPC from 5% input of pre-immunoprecipitation sample showed three bands which represents the three glycosylation states of PrPC; di-glycosylated (di), mono-glycosylated (mono), and un-glycosylated form (un), respectively). (E) Densitometric quantification of total PrPC and Caspr bound PrPC. (F) Densitometric quantification of di-glycosylated PrPC, (G) unglycosylated PrPC. (H) Representative immunoblots for βAPP, and Actin loading control. (I) Densitometric quantification of βAPP and Actin (*P < 0.05, n = 4 for both genotypes). (J) Immunofluorescent images showed axonal PrPC and co-localisation with Caspr within LSSC white matter (WM) of ngr1+/+ whereas, lack of axonal PrPC staining in WM was found in ngr1-/- mice. PrPC expression in motor neuronal soma within the gray matter (GM) of LSSC was found in both genotypes. Illustration showing where the WM and GM images were taken from LSSCs are shown at the right-hand side (WM scale bars = 10 μm; GM scale bars = 50 μm). (K) In wild-type, paranodal junction is tightly organised by the putative interaction among glial Neurofascin 155, axonal Caspr and Contactin. Furthermore, tight interaction between Caspr and PrPC inhibits Reelin-mediated Caspr cleavage, which allows for tight compaction of paranodal loops and myelin sheath. In the absence of NgR1, reduced interaction between PrPC and Caspr mediates Reelin-mediated cleavage of Caspr, which leads to de-compaction of paranodal loops and myelin sheath. Furthermore, paranodal localisation of Nogo-A in ngr1-/- whereas broad expression of Nogo-A throughout the axo-glial unit is found in wild-type.

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