- Guinea pig polyclonal
- Suitable for ICC/IF, IHC and WB
- Reacts with human
| Quantity | 100 µl |
|---|---|
| Antibody Type | Polyclonal |
| Host | Guinea pig |
| Conjugate | Unconjugated |
| Application | ICC/IF, IHC, WB |
| Purification | Stabilized antiserum |
| Reactivity | Human |
| Storage | Short term at 2-8°C; long term storage in aliquots at -20°C; avoid freeze/thaw cycles |
| Intended use | Research use only |
| Immunogen | Recombinant human keratin K17 |
| Formulation | Contains 0.09% sodium azide and 0.5% BSA |
| UniprotID | Q04695 (Human) |
| Synonym | Keratin, type I cytoskeletal 17, 39.1, Cytokeratin-17, CK-17, Keratin-17, K17, KRT17 |
| Note | Centrifuge prior to opening |
| Tested applications | Tested dilutions |
|---|---|
| Immunocytochemistry (ICC)/ Immunofluorescence (IF) | 1:100-1:200 |
| Immunohistochemistry (IHC) - frozen | 1:100-1:200 |
| Immunohistochemistry (IHC) - paraffin | 1:50 (microwave treatment recommended) |
| Western Blot (WB) | 1:5,000-1:10,000 |
The antiserum represents an excellent marker to distinguish myoepithelial cells (positive for keratin K17) from luminal epithelium of various glands (mammary, sweat, salivary, bronchial, tracheal, laryngeal, esophageal) and benign from malignant forms of e.g. mammary gland tumors. In the epidermis suprabasal staining is found only in cornifying regions and in the outer root sheath of hair follicles.
- PVDF membranes show better results than nitrocellulose (higher capacity, allows for more stringent washing conditions in case of background problems).
- Use freshly prepared blocking solution (e.g. 5% nonfat dry milk, 0.05% Tween 20), block for at least 1 h at room temperature.
- Use the antibody in a higher dilution, but prolong incubation time and exposure time.
- Always use a fresh aliquot of the antibody.
- Do not repeatedly freeze the antibody (eventually centrifuge shortly after thawing to remove cryo-precipitates).
- Include an additional washing step.
You might also try more stringent wash conditions, e.g. add 0.5 M NaCl to the wash buffer. - Always use a fresh aliquot of secondary antibody.
- In case you use ECL most the guinea pig antibody should be diluted further in order to get rid of the background.
The supernatant format contains FCS proteins from cell culture medium supplemented with FCS.
The serum antibodies contain other proteins present in serum.
Most of our liquid antibodies and reconstituted lyophilized antibodies may be stored for short term storage (up to 3 month) at 2-8°C. For long term storage we recommend to store the antibody at -20°C in aliquots. Please avoid freeze and thaw cycles.
Most of our conjugated antibodies should be stored at 2-8°C.
The individual storage conditions are mentioned on the datasheet.
Positive tissue: outer root sheath of hair follicle, sweat gland epithelium on the foot pads, filiform papillae of tongue.
- Homogenization (e.g. with polytron) of tissue samples in buffer L (140 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10 mM Tris-HCl pH7.6) supplemented with protease inhibitors; use 1 ml buffer for approx. 0.1 g tissue.
- To reduce viscosity by high DNA contents, benzonase treatment can be included (30 min at 37°C).
- Centrifugation for 10 min (max. speed, table top centrifuge).
- Resuspend pellet in "Triton extraction buffer" (buffer L plus 1% Triton X-100) using e.g. a dounce homogenizer.
- Centrifugation for 10 min (max. speed, table top centrifuge).
- Resuspend pellet in "high salt extraction buffer" (buffer L plus 1.5 M KCl) using e.g. a dounce homogenizer.
- Centrifugation for 10 min (max. speed, table top centrifuge).
- Resuspend pellet in "Triton extraction buffer" (buffer L plus 1% Triton X-100) using e.g. a dounce homogenizer.
- Centrifugation for 10 min (max. speed, table top centrifuge).
- Resuspend pellet in Laemmli buffer and boil (5 min) for SDS-PAGE.
- Methanol/ acetone fixation: Immerse slide in precooled (-20°C) methanol for 5 min, immerse in precooled (-20°C) acetone for 30-60 sec, let specimen air dry before antibody incubation.
- Methanol/ acetone fixation plus detergent permeabilization: After methanol/ acetone fixation and air-drying dip slide either in a solution containing 0.1-0.2% Triton X-100 in PBS or in 0.1% saponin in PBS for 1-5 min at room temperature (enhances accessibility of many cytoskeletal antigens).
- Air-drying of the section.
- Block with the serum of the species in which the secondary antibody was raised for 30 min.
- Incubation with 1st antibody 1 h at RT in moist chamber.
- Wash 3x with PBS.
- Incubation with appropriate fluorescent secondary antibody, 30-60 min at RT.
- Wash 3x with PBS.
- Immerse shortly into ethanol.
- Let air dry.
- Cover with mounting medium.
In guinea pigs the antibody concentration in serum varies from 10 to 20 mg/ml; specific antibodies represent normally about 0.1-1% of total IgG. Total protein concentration varies from 40 to 65 mg/ml, with the main constituent (about 60%) being albumin.
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