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Immunofluorescence analysis of Histidine tag (6XHis) was done on HEK-293 cells transiently overexpressing His- IGFBP6. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Histidine (C-term) tag (6XHis) Mouse Monoclonal Antibody (R93025 ) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Rabbit Anti-Mouse IgG Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938).F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing cytoplasmic localization. Panel e is untransfected HEK-293 cells. The images were captured using a Nikon microscope at 20X magnification.
|Tested species reactivity||Tag|
|Host / Isotype||Mouse / IgG2b|
|Immunogen||6x His synthetic peptide|
|Contains||0.01% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay Dependent|
|Flow Cytometry (Flow)||1:100-1:500|
|Immunoprecipitation (IP)||Assay Dependent|
|Western Blot (WB)||1:1000-1:5000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
NOTE: R930-25 was previously sold under product #46-0693. When re-ordering, please use #R930-25.
This antibody can be used to detect expression of C-terminal His fusion proteins from bacterial, insect, and mammalian cells.
This product contains enough material for 25 Western blots. It is functionally tested using 500ng of an E. coli expressed fusion protein containing a C-terminal polyhistidine (6xHis) epitope. A signal can be detected when tested against 100ng of recombinant Positope™ protein. For Western blot, dilute in PBS containing 0.05% Tween-20 and 5% nonfat, dry milk (PBSTM).
This product has also been used successfully to immunoprecipitate fusion proteins that contain the 6x His tag.
Using chemiluminescence as the detection method, no cross-reactivity has been observed in bacterial lysates. In mammalian lysates, a few cross-reactive proteins have been observed upon overexposure of blots.
Epitope tags provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. Expression of recombinant proteins in E. coli as a fusion protein with neighboring histidine residues is one of the most popular methods of epitope tags. The affinity of the histidine-tag motif to Ni2+ by chelation is strong and selective enough to enable purification of the protein to homogeneity by affinity chromatography on a Ni2+-NTA adsorbent.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
46-0693; 460693; His Tag; Histidine Epitope Tag; Histidine Tag; R930-25; R93025