Application
Western blot
Sample
Human Cell lysate - whole cell (human HEK293 cells over-expressing)
Loading amount
150 µg
Specification
human HEK293 cells over-expressing
Gel Running Conditions
Reduced Denaturing (denature at 70C for 10mins, 7.5% gel)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Other product details
Dilution
1/25
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: overnight
Secondary antibody
Name
Non-Abcam antibody was used: goat anti-mouse IgG
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/5000
Detection
Detection method
ECL+
Exposure
1 hour(s), 0 minute(s) and 0 second(s)
Bands
Specific: none kDa Non-specific: 150, 120, 100, 60, 50 kDa
Positive control
HEK293 cells transfected with OATP1B3 plasmid purchased from Genecopoeia (EX-U0996-M02), transporter function was measure by 3H-estrone-3-sulfate uptake. As to the literature, the expected bands should be ~85KDa and ~120KDa when expressing in the same cell line. (Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8). Letschert K, Keppler D, König J. Pharmacogenetics. 2004 Jul14(7):441-52.)
Negative control
HEK293 parental cells (labeled as "NC" in the image)
Additional data
Additional Notes
anti-SLCO1B3 did not give a specific band in correct location when tested on transfected HEK293 overexpressing SLCO1B3 and non transfected HEK293 (negative control), therefore I rated the antibody as poor
Abcam response
Thank you for your honest feedback. This antibody has not yet been tested by the lab in WB. The expected band of the unmodified protein would be at about 76 kDa - according to the database entry Q9Y6L6 (UniProt). Glycosylation can shift the MW higher. Suggestions that could help to reduce the background are: loading less protein (e.g. 20-30 ug), using less primary antibody (e.g. 1/100 or 1/500) and reducing the incubation time with the antibody (e.g. 2h at RT). It could be that the antibody is not suitable for WB, but we would welcome additional feedback from other customers.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Abcam user community
Verified customer
投稿 Sep 08 2011