Specific protocols
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To our knowledge, customised protocols are not required for this product.
Please try the standard protocols listed below and let us know how you get on. |
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General protocols
Useful resources:Western blotting (WB) protocols:Immunohistochemistry (IHC) / Immunocytochemistry (ICC) protocols:Chromatin Immunoprecipitation (ChIP) protocols:Dot blot protocols:ELISA protocols:ELISPOT protocols: Flow cytometry / FACS protocols:Immunoprecipitation (IP) protocols: |
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
Immunocytochemistry/ Immunofluorescence - FGF9 antibody (ab71395)
ICC/IF image of ab71395 stained MCF7 cells. The cells were 4% PFA fixed (10 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 (ab71395, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa, hek293 and MCF7 cells at 5µg/ml.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - FGF9 antibody (ab71395)
IHC image of FGF9 staining in FFPE human kidney carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab71395, 1µg/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.
Western blot - FGF9 antibody (ab71395)
All lanes : Anti-FGF9 antibody (ab71395) at 1 µg/ml
Lane 1 : Brain (Human) Tissue Lysate - adult normal tissue (ab29466)
Lane 2 : Stomach (Human) Whole Cell Lysate - tumor tissue (ab29678)
Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 4 : Brain (Human) Tissue Lysate - adult normal tissue (ab29466) with FGF9 peptide (ab86940) at 1 µg/ml
Lane 5 : Stomach (Human) Whole Cell Lysate - tumor tissue (ab29678) with FGF9 peptide (ab86940) at 1 µg/ml
Lane 6 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with FGF9 peptide (ab86940) at 1 µg/ml
Lysates/proteins at 10 µg per lane.
Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Predicted band size : 23 kDa
Observed band size : 50 kDa (why is the actual band size different from the predicted?)
Additional bands at : 100 kDa. We are unsure as to the identity of these extra bands.
The peptide blocking data above shows that ab71395 detects a protein of approximately 50 kDa, thought to be FGF9. It is possible that this band is significantly higher than expected due to modifications at potential glycosylation sites.
Western blot - Anti-FGF9 antibody (ab71395)
All lanes : Anti-FGF9 antibody (ab71395) at 1 µg/ml
Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Kidney (Mouse) Tissue Lysate
Lysates/proteins at 10 µg per lane.
Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 23 kDa
Observed band size : 55 kDa (why is the actual band size different from the predicted?)
Additional bands at : 105 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 2 minutes
FGF9 antibody for Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) in Human (71395)
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