All lanes : Anti-PFKFB3 antibody (ab96699) at 1/1000 dilutionLane 1 : A431 whole cell lysateLane 2 : H1299 whole cell lysateLysates/proteins at 30 µg per lane.
![PFKFB3 was immunoprecipitated using 0.5mg A431 whole cell extract, 5µg of Rabbit polyclonal to PFKFB3 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, A431 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 ab96699.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 70kDa: PFKFB3.](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_4/10376_PFKFB3-Primary-antibodies-ab96699-4.jpg)
PFKFB3 was immunoprecipitated using 0.5mg A431 whole cell extract, 5µg of Rabbit polyclonal to PFKFB3 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, A431 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 ab96699.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 70kDa: PFKFB3.
IHC image of PFKFB3 staining in Human breast fibroadenoma formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab96699, 5µg/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.For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.