![Cystatin C was immunoprecipitated using 0.5mg Caco2 whole cell extract, 5µg of Rabbit polyclonal to Cystatin C 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, Caco2 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 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab68290.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 14kDa; Cystatin C](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_2/4569_ab68290-195134-IPV021ab6829012min.jpg)
Cystatin C was immunoprecipitated using 0.5mg Caco2 whole cell extract, 5µg of Rabbit polyclonal to Cystatin C 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, Caco2 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 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab68290.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 14kDa; Cystatin C
All lanes : Anti-Cystatin C antibody (ab68290) at 1 µg/mlLane 1 : Caco 2 (Human colonic carcinoma cell line) Whole Cell LysateLane 2 : Spinal Cord (Human) Tissue Lysate - adult normal tissue (ab29188)Lane 3 : U2OS (Human osteosarcoma cell line) Whole Cell LysateLysates/proteins at 10 µg per lane.SecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ICC/IF image of ab68290 stained HeLa 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 (ab68290, 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) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
IHC image of Cystatin C staining in Human Liver Cancer FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab68290, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX
developed using the ECL techniquePerformed under reducing conditions.