All lanes : Anti-Alpha Skeletal Muscle Actin antibody (ab52218) at 1/500 dilutionLane 1 : extracts from LOVO cells,Lane 2 : extracts from LOVO cells, with immunizing peptide
Ab52218 staining human skeletal muscle tissue. Staining is localised to cytoplasm.Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
![Skeletal Muscle Actin was immunoprecipitated using 0.5mg Mouse Skeletal Muscle tissue lysate, 5µg of Rabbit polyclonal to Skeletal Muscle Actin 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, Mouse Skeletal Muscle tissue lysate 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 ab52218.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to anti-rabbit HRP (IgG light chain) (ab99697).Band: 45kDa; Skeletal Muscle Actin](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_1/5812_skeletal-muscle-Actin-Primary-antibodies-ab52218-5.jpg)
Skeletal Muscle Actin was immunoprecipitated using 0.5mg Mouse Skeletal Muscle tissue lysate, 5µg of Rabbit polyclonal to Skeletal Muscle Actin 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, Mouse Skeletal Muscle tissue lysate 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 ab52218.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to anti-rabbit HRP (IgG light chain) (ab99697).Band: 45kDa; Skeletal Muscle Actin