- Guinea pig polyclonal
- Suitable for IHC and WB
- Reacts with bovine, human and mouse
| Quantity | 100 µl |
|---|---|
| Antibody Type | Polyclonal |
| Host | Guinea pig |
| Conjugate | Unconjugated |
| Application | IHC, WB |
| Purification | Stabilized antiserum |
| Reactivity | Bovine, Human, Mouse |
| 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 K5 |
| Formulation | Contains 0.09% sodium azide and 0.5% BSA |
| UniprotID | Q5XQN5 (Bovine), Q7RTS7 (Human), Q922U2 (Mouse) |
| Synonym | Keratin, type II cytoskeletal 74, Cytokeratin-74, CK-74, Keratin-5c, K5C, Keratin-74, K74, Type II inner root sheath-specific keratin-K6irs4, Type-II keratin Kb37, KRT74, K6IRS4, KB37, KRT5C, KRT6IRS4 |
| Note | Centrifuge prior to opening |
| Tested applications | Tested dilutions |
|---|---|
| Immunohistochemistry (IHC) - frozen | 1:100 |
| Immunohistochemistry (IHC) - paraffin | 1:50 (microwave treatment recommended) |
| Western Blot (WB) | 1:1,500-1:5,000 |
Basic keratin K5 (Mr 58,000; formerly also designated cytokeratin 5), expressed in basal and first suprabasal layers of epidermis. Highly specific staining pattern in human and murine skin sections.
Tumors Specifically Detected: The antiserum reacts with keratin K5, expressed in the basal cells of the larynx, esophagus, trachea, bladder, cervix, vagina, breast acini, skin and sweat glands.
In several studies the correlation between the expression of keratin K5 in different types of carcinomas (putatively derived from basal epithelial cells) and prognosis has been discussed (see publications).
- 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.
- Esophagus
- Lacatating mammary gland
- Urinary bladder
- Sweet glands
- 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.
- Mild fixation with PFA (e.g. 2% PFA) is recommended.
- Preincubation with 0.1-0.2% Triton-X-100 in PBS prior to fixation is optional and can in some cases be recommended for cytoskeletal antigens when the epitope is not readily accessible.
- After PFA fixation, rinse with 50 mM ammonium chloride.
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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