Antibodies FAQs
We offer sample sizes for some of our antibodies.
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.
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.
We offer bulk amounts starting from 1 mg for many of our mouse monoclonal or recombinant antibodies. Please contact us for further information or use the “bulk button” on the product page.
Most of our purified mouse antibodies contain 0.5% BSA as stabilizer. If BSA was added to the antibody solution, it is stated in the datasheet.
The supernatant format contains FCS proteins from cell culture medium supplemented with FCS.
The serum antibodies contain other proteins present in serum.
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 antibodies contain 0.09% sodium azide as preservative. If a preservative is added, it is mentioned in the datasheet.
PROGEN antibodies are shipped at ambient temperature. The antibodies are stable at ambient temperature for the shipment period. Please store the antibodies as indicated in the datasheet upon arrival.
The optimal antibody dilution for your specific protocol and application needs to be titrated in your lab with your equipment and sample. The optimal dilution may vary between protocols and samples. A good dilution for starting the titration is the dilution mentioned in the datasheet. If the sample needs a specific treatment (eg. Antigen retrieval for IHC on FFPE sections) this should also be mentioned on the antibody datasheet.
Please find the solvent for reconstitution and the amount of liquid for reconstitution on the antibody datasheet. The buffer formulation after reconstitution is also mentioned in the datasheet.
Lyophilized antibodies can be stored at 2-8°C until expiration.
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.
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 unpurified supernatant, ascites, unpurified guinea pig serum and unpurified rabbit serum is not determined.
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 support@progen.com.
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 support@progen.com.
The concentration of specific antibody in our guinea pig serum is not determined.
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.
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.
PROGEN offers different formats for mouse monoclonal antibodies. Different antibodies may not be offered in each format, the following are all possible formats:
- 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.
The expiration date of our antibodies is indicated on the product label.
Protocols for different applications can be found in the downloads tap on the antibody product page or on the following page: https://www.progen.com/Support/Downloads/
Publications citing PROGEN antibodies are available on the antibody product page in the Reference tab.
If the antigen retrieval buffer is not mentioned in the datasheet, the optimal buffer needs to be tested.
- 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).
Phage Display FAQs
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.
After reconstitution, the hyperphage can be kept at 2-8°C for several days, however, the infection potential will decrease gradually.
Hyperphage stored at 2-8°C for several weeks (up to 2 months) were still active, however, the pfu was more than 50% decreased.
For long term storage it is possible to add 20% glycerol and store the hyperphage at -80°C. This will also lead to a decrease in activity. Hyperphage stored at 2-8°C for several weeks (up to 2 months) were still active, however, the pfu was more than 50% decreased.
The concentration of our hyperphage is lot dependent. We try to keep the concentration > 1E+12 particles/ml. The specific concentration is indicated on the product label. Please contact us to ask for the concentration of your specific lot or the current lot in stock.
10 mM Tris-HCl (pH 7.5), 20 mM NaCl, 2 mM Na-EDTA
Please be informed that the packaging cell line has been patented (i.e. it is proprietary) and is unfortunately not available publicly.
Therefore it is not possible to amplify hyperphage in your lab.
Therefore it is not possible to amplify hyperphage in your lab.
TG1 is compatible with hyperphage and the classical strain for phage display. It is very suitable for panning (one panning round per day).
For the last panning round XL1-Blue MRF’ may be used, since the plasmid stability, quality and yield is higher with this strain.
Alternatively to TG1 or XL1-Blue MRF’, ER2783 is also suitable for library cloning and packaging. For the last panning round XL1-Blue MRF’ may be used, since the plasmid stability, quality and yield is higher with this strain.
It is important to use a bacterial strain with lacIq genotype (for example XL1 Blue or TOP10F’) with our pSEX81 plasmid. Bacterial strains with lacIq genotype carry the lacIq repressor that represses expression from the lac promoter and so allows cloning of toxic genes. The PIII fusion protein expressed by our pSEX81 vector is toxic to the bacterial cells.
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.
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.
Only vectors coding for a fully functional PIII protein are suitable for the hyperphage system, for example pSEX81.
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/).