All lanes : Anti-IRAK antibody (ab238) at 1/2000 dilutionLane 1 : THP-1 (THP)whole cell lysateLane 2 : HeLa (HL) whole cell lysate
ab238 at 10µg/ml staining Hela cells by ICC/IF
IHC image of ab238 staining in human breast carcinoma 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 ab238, 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.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.
ab238 staining IRAK in Human platelet cells by Flow cytometry.Cells were fixed in paraformaldehyde and permeabilized using 0.1% Triton-X-100 in 2% BSA for 15 minutes. Primary antibody used at a 1/200 dilution and incubated for 16 hours at 4°C. The secondary antibody used was an Alexa Fluor®488 conjugated chicken anti-rabbit IgG (H+L) at a 1/500 dilution. P : Permeabilized US : Unstained (Red Peak) IGG RB : IgG Rabbit (Blue Peak) IRAK Ab (Green Peak)See Abreview
ab238 staining IRAK in murine RAW 264.7 cells by Immunocytochemistry/ Immunofluorescence.Cells were fixed in paraformaldehyde, permeabilized using 0.1% Triton-X100 in 2% BSA for 15 minutes, blocked with 2% BSA for 1 hour at 22°C and then incubated with ab238 at a 1/150 dilution for 16 hours at 4°C. The secondary used was an Alexa-Fluor 488 conjugated chicken anti-rabbit IgG (H+L) used at a 1/1000 dilution.See Abreview
![IRAK was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to IRAK 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 ab238.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 80kDa: IRAK](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_3/9796_IRAK-Primary-antibodies-ab238-22.jpg)
IRAK was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to IRAK 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 ab238.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 80kDa: IRAK
Immunofluorescence of IRAK in HeLa cells using ab238 at 20 ug/ml.