Anti-Hippocalcin antibody (ab24560)

Western blot - Hippocalcin antibody (ab24560)

All lanes : Anti-Hippocalcin antibody (ab24560) at 1 µg/ml

Lane 1 : Mouse Brain
Lane 2 : Day 0 Mouse Brain

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 23 kDa
Observed band size : 23 kDa
Additional bands at : 14 kDa (possible cleavage fragment),45&52&100 kDa (possible cross reactivity).

Immunohistochemistry (PFA fixed) - Hippocalcin antibody (ab24560)

Immuofluorescent staining for Hippocalcin in the rat hippocampus using Rabbit polyclonal to Hippocalcin (ab24560). This figure is a montage of pictures acquired with a X10 objective and shows expected abundance of staining in parts of the hippocampus such as the CA1 and CA3 and an absence of staining (as expected) in the cortex and the corpus callosum. ab24560 was used at 1/1000 (0.2µg/ml) incubated overnight at room temperature. Secondary antibody used was anti-rabbit Alexa Fluor 488 at 1/1000 incubated for 2 hours at room temperature. Rat brain tissue was perfusion fixed with 4% PFA followed by overnight post-fixation in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. 30µm coronal sections were cut on a cyrostat and immunohistochemistry performed by the 'free floating' technique.

Sophie Pezet, King`s college London, United Kingdom

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Hippocalcin antibody (ab24560)

Image courtesy of Human Protein Atlas

ab24560 staining Hippocalcin in human cerebellum. Paraffin embedded tissue was cut into 4µm sections and incubated with ab24560 (1/350 dilution) for 30 minutes at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab24560 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

Immunoprecipitation - Anti-Hippocalcin antibody (ab24560)

Hippocalcin was immunoprecipitated using 0.5mg Mouse Brain whole tissue lysate, 5µg of Rabbit polyclonal to Hippocalcin 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 ab24560.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 23kDa: Hippocalcin; non specific - 60kDa: We are unsure as to the identity of this extra band.

Western blot - Anti-Hippocalcin antibody (ab24560)

All lanes : Anti-Hippocalcin antibody (ab24560) at 1 µg/ml

Lane 1 : Marker
Lane 2 : Zebrafish brain homogenate (20ug)
Lane 3 : Mouse brain homogenate (20ug)

Secondary
Goat polyclonal to Rabbit IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 23 kDa
Observed band size : 23 kDa


Exposure time : 1 minute

Immunocytochemistry/ Immunofluorescence - Anti-Hippocalcin antibody (ab24560)

ICC/IF image of ab24560 stained SKNSH cells. The cells were 4% formaldehyde 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 ab24560 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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) at a concentration of 1.43µM.