anti-Plectin guinea pig polyclonal, serum
- Guinea pig polyclonal
- Suitable for ICC/IF, IHC and WB
- Reacts with bovine, human and mouse
|Application||ICC/IF, IHC, WB|
|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||C-terminal “C” domain of human recombinant Plectin (aa 4367 - 4684)|
|Formulation||Contains 0.09% sodium azide and 0.5% BSA|
|UniprotID||A0A3Q1M6Q7 (Bovine),Q15149 (Human),Q9QXS1 (Mouse)|
|Synonym||Plectin, PCN, PLTN, Hemidesmosomal protein 1, HD1, Plectin-1, PLEC, PLEC1|
|Note||Centrifuge prior to opening|
|Tested applications||Tested dilutions|
|Immunocytochemistry (ICC)/ Immunofluorescence (IF)||Assay dependent|
|Immunohistochemistry (IHC) - frozen||1:500|
|Immunohistochemistry (IHC) - paraffin||1:500 (microwave treatment recommended)|
|Western Blot (WB)||1:16,000|
Calculated MW 532,000 protein plectin, localizing to plasma membrane attachment sites of intermediate filaments and microfilaments (actin) such as desmosomes and hemidesmosomes, Z-line structures and dense plaques of striated and smooth muscle. Strong positive reaction is also found with endothelia, fibro-blasts and infiltrating cells (e.g. lymphocytes). Human glia cells are specifically detected.
Reactivity on cultured cell lines: HL-60, Huvec, BMGE, 3T3
Q & A's
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- 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.
- 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.
- Use wet transfer instead of semidry method.
- Reduce the amount of methanol in the transfer buffer (< 20%).
- Increase the transfer time (e.g. on at 100-200 mA).
- If the protein is low abundant (e.g. nephrin expressed only in podocytes) enrich your protein of interest.
- Pretreatment of the sample with DNase.
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.