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Our Abpromise guarantee covers the use of ab40084 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 1µg for 106 cells.
Methanol or paraformaldehyde fixed cells.
ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
|ICC/IF||Use a concentration of 1 - 5 µg/ml.|
|IHC-FoFr||Use at an assay dependent concentration. PubMed: 21688176|
|WB||1/5000. Detects a band of approximately 55 kDa (predicted molecular weight: 54 kDa).
Previous lots of this antibody gave good results in WB as published in Abreviews. We however are observing multiple bands with recent lots. GLUT1 is a multi-pass membrane protein so we can recommend heating the samples 60 - 70C for 10 -15 minutes instead of boiling in sample buffer. We will welcome any feedback the successful users have
|IHC-P||1/200. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
Immunohistochemical analysis of 10% buffered formalin-fixed paraffin-embedded human dermal carcinoma tissue sections, labelling GLUT1 with ab40084 at a dilution of 1/100 incubated for 12 hours at 4°C. Heat mediated antigen retrival was performed with 10mM sodium citrate buffer at pH 6.0. Blocking was with 5% serum incubated for 1 hour at 21°C. The secondary was a Donkey anti-mouse polyclonal Alexa Fluor® 647 conjugate at 1/200. Counterstaining is DAPI in blue against Nuclear DNA.
Overlay histogram showing HeLa cells stained with ab40084 (red line). The cells were fixed with methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab40084, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/250 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 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) used under the same conditions.
ab40084 staining Glucose Transporter GLUT1 in mouse brain tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde and blocked with 20% serum for 20 minutes at room temperature. The sample was incubated with primary antibody (1/200) . A Biotin-conjugated donkey anti-mouse polyclonal (1/200) was used as the secondary antibody.
4oC (1 freeze/thaw)
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