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Full length native protein (purified) (S. cerevisiae).
This antibody can be used as a loading control on Western blots (Allen et al.) and is not detected by anti-mouse Ig secondaries. It has been used in epitope tagging procedures to detect proteins tagged with a C-terminal Gly-Gly-Phe(OH) epitope. Under some circumstances this antibody may cross-react with other protein including E. coli rec A and oxidized actin.
Our Abpromise guarantee covers the use of ab6160 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ELISA||Use at an assay dependent dilution.|
|IHC-Fr||Use at an assay dependent dilution.|
|IP||Use at an assay dependent dilution.|
|RIA||Use at an assay dependent dilution.|
|WB||1/5000 - 1/10000.|
|Flow Cyt||Use 1µg for 106 cells.|
|ICC/IF||1/1000. (see PMID: 16230461)|
|IHC (PFA fixed)||Use a concentration of 5 µg/ml. (from PubMed:16966421)|
|IHC-P||Use at an assay dependent dilution.|
|IHC (Methanol fixed)||1/100. (from PubMed:16943269).|
IHC image of Tubulin staining in human colon formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 ab6160, 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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
ICC/IF image of ab6160 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab6160 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rat (ab150165) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot against tubulin with ab6160. Secondary Rabbit polyclonal to Rat IgG H&L (HRP) (ab6734) was used at 1/2000.
Exposure time: 10 seconds.
Lane 1-2: 20
Lane 3-4: 20
Lane 1,3: ab6160 at 1/5000.
Lane 2,4: ab6160 at 1/10000.
ab6160 staining Tubulin in mouse MEF cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with 2% PFA and 96% Ethanol. Samples were incubated with primary antibody (1/2000 in 0.1% Saponin/1% BSA/PBS) for 1 hour. A Cyt3®-conjugated goat polyclonal to rat IgG (H&L) was used at dilution at 1/500 as secondary antibody. Red staining in the image represents Tubulin, whereas the green one resembles gamma-tubulin.
ab6160 staining mouse prostate tissue sections by IHC-P. The tissue was serial sectioned at 6 microns, formaldehyde fixed and subjected to heat mediated antigen retrieval prior to blocking in 3% peroxidase for 5 minutes at 27°C. The primary antibody was diluted 1/500 and incubated with the sample for 16 hours at 4°C. A Cy5® conjugated goat anti-rat antibody was used as the secondary.
ab6160 at 1/1000 dilution staining Tubulin in human WBC cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in acetone and then blocked in 5% serum for 1 hour at 25°C. No permeabilization was done. The primary antibody was used at 1/1000 dilution in PBS-Tween and incubated with sample at 4°C for 16 hours. An Alexa Fluor® 594 conjugated goat polyclonal to rat IgG was used as secondary at 1/500 dilution.
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