For our lyophilized antibodies we offer 10 µg lyophilized samples and for our liquid antibodies we offer 200 µl liquid purified antibodies. The available formats can be found on the antibody product page.
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.
- 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 concentration of purified antibodies is mentioned on the datasheet.
For prediluted antibodies the concentration may vary from lot to lot. The concentration of these antibodies is not mentioned on the datasheet and can be requested at firstname.lastname@example.org.
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.
- Supernatant and supernatant concentrate: This format contains hybridoma cell culture supernatant. The antibody is not purified and the antibody concentration is not determined. The antibody concentration may vary from lot to lot. Therefore we recommend to titrate the optimal concentration for the application used for each new lot.
- Lyophilized, purified: This format contains purified antibody in lyophilized form. The reconstitution of this antibody is described in the datasheet. The buffer composition after reconstitution is also mentioned on the datasheet.
- Liquid, purified: This format contains purified antibody in liquid format. The concentration is mentioned on the datasheet.
- Prediluted, purified: This format contains purified antibody in liquid format. Most antibodies in this format are diluted to be ready-to-use for IHC with standard tissue. But some antibodies of this format need further dilution for IHC. This is mentioned on the datasheet.
- An acidic retrieval solution based on citric acid buffer is often used for cytoskeletal and membrane antigens.
- A high pH retrieval solution is often suitable for nuclear antigens present in low amounts (like p53 antigen).
You can perform 8 PCR reactions with each primer pair.
The Mouse IgG Library Primer Set contains 50 primers in 2 primer sets. The primers are ready-to-use for common PCR (conc. 10 pmol/µl in 10 mM Tris-HCl pH 8, 1 mM sodium EDTA).
With the first set of primers 22 separate reactions should be performed. Each variable heavy chain forward primer (1A-L) has to be combined with the constant region IgG reverse primer (1M). Analogous, each κ- and λ-light chain forward primer (1N-W, 1Y) has to be combined with the corresponding constant region reverse primer (1X or 1Z), respectively.
With the second primer set 22 separate reactions should be performed to add the restriction sites for cloning into the pSEX81 vector.
Hyperphage stored at 2-8°C for several weeks (up to 2 months) were still active, however, the pfu was more than 50% decreased.
Therefore it is not possible to amplify hyperphage in your lab.
For the last panning round XL1-Blue MRF’ may be used, since the plasmid stability, quality and yield is higher with this strain.
DH5alpha or TOP10 (without F episome) are lacIq negative and therefore the toxic proteins are expressed in these cells. When using lacIq negative cell lines with the pSEX81 vector you will receive only a few colonies and most often the plasmid is somehow mutated in these colonies.
We also recommend to include 100 mM Glucose to the culture medium to avoid possible mutations of the plasmid.
According to the literature pCANTAB vector is compatible with hyperphage, although with lower efficiency as for example pSEX81 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1866265/).