This antibody detects a band at approximately 88kDa that corresponds in size to ZW10. It also detects bands at 75 and 45kDa that may be degradation products or crossreactivity. All of these bands are competed away by the addition of the immunizing peptide, suggesting that they are specific interactions.
ZW10 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to ZW10 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, Jurkat whole cell extract 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 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab21582. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 88kDa: ZW10
Immunocytochemistry/ Immunofluorescence - Anti-ZW10 antibody (ab21582)This image is courtesy of Scott Slattery and Mike Mancini
Panel one shows staining of HeLa cells by anti-ZW10 (ab21582) in green and DAPI in blue. The second panel shows staining by anti-ZW10 in green and SH-CREST in red, which stains the centromeres. In most cells at interphase, ZW10 is present diffusely in the cytoplasm. In prometaphase, it is associated with the kinetochore. Fix cells for 30 minutes on ice in 4% formaldehyde in PEM.
Quench autofluorescence 2 x 5 minutes with 1 mg/ml Na borohydride or 100 mM ammonium chloride in PEM.
Permeablize 30 minutes with 0.5% TX-100 in PEM.
Block 30 minutes in 5% milk in TBST.
Primary antibody incubated overnight at 4oC diluted 1/100 in 5% milk in TBST.
Secondary antibody 1 hour at RT diluted in 5% milk in TBST.
Post-fix 20 min. on ice in 4% formaldehyde in PEM.
Quench autofluorescence 2 x 5 minutes with ammonium chloride in PEM.
Counterstain with DAPI in TBST.
Mount with ProLong Gold antifade reagent from Invitrogen.
Notes: Ample washing between each step. TBST = Tris buffered saline +
Image courtesy of Human Protein Atlasab215820 staining ZW10 in Human stomach, showing a strong staining pattern in gastric pits with lower levels in the basement membrane. Paraffin embedded human stomach tissue was incubated with ab21582 (1/150 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.
ab21582 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
Immunocytochemistry/ Immunofluorescence - Anti-ZW10 antibody (ab21582)This image is courtesy of an Abreview submitted by Dr Kirk McManus
ab21582 (1/200) staining ZW10 in asynchronous mouse 10T1/2 cells (green). Cells were pre-extracted with PEM (1min) at room temperature, fixed in paraformaldehyde (10 mins), washed in KB+ buffer and counterstained with DAPI in order to highlight the DNA/nucleus (red). Please note that ab21582 might only perform well when using this specific protocol. Please refer to abreview for further experimental details.