Lane 1 : Lanes 2 - 6 : Anti-RAD54 antibody (ab10705) at 1/500 dilutionLane 1 : MarkerLane 2 : HeLa nuclear cell lysateLane 3 : HeLa cell lysateLane 4 : A432 cell lysateLane 5 : MCF7 cell lysateLane 6 : Hek 293 cell lysateSecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab6721)
IHC image of ab10705 staining in Human breast adenocarcinoma 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 ab10705, 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.
![RAD54 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to RAD54 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, Hela 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 ab10705.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 70kDa; RAD54, non specific band: 75 kDa, We are unsure as to the identity of this extra band. 99kDa band:due to background seen in No ab control lane (2).](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_4/20156_RAD54-Primary-antibodies-ab10705-4.jpg)
RAD54 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to RAD54 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, Hela 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 ab10705.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 70kDa; RAD54, non specific band: 75 kDa, We are unsure as to the identity of this extra band. 99kDa band:due to background seen in No ab control lane (2).