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Fusion protein (GST-tag) corresponding to Human KMT5A/ SETD8/ Pr-SET7 (N terminal).
Our Abpromise guarantee covers the use of ab3798 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||1/10 - 1/50.|
|WB||1/1000. Predicted molecular weight: 42.89 kDa.|
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|Flow Cyt||Use 1µg for 106 cells.|
Immunofluoresence was carried out using mouse cells in culture. Conditions: 4% PFA fixation and dilution with 0.5% triton, 10mg/ml BSA in PBS. Dilution of ab3798 used: 1/10. Green = ab3798 detected with secondary antibody (Alexa 488). Blue = DAPI stain.
ab3798 (1µg/ml) staining SETD8 in Human thyroid gland using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining of epithelial cells of the thyroid follicle.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min 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, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
Overlay histogram showing HeLa cells stained with ab3798 (red line). The cells were fixed with 100% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab3798, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.