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Product Form Descriptions
Supernatant is serum-free hybridoma conditioned culture media containing bovine serum albumin for protein stabilization (HB101 medium).
Concentrate is the retentate (~10mls) after ultrafiltration of 100mls of supernatant spun through a 30kD molecular exclusion filter.
Ascites is the accumulation of excess fluid due to growth of hybridomas after injection of the cells into the peritoneal space of a mouse. Ascites fluid will contain highly concentrated monoclonal antibody along with other mouse proteins. The Ascites product available from DSHB has been centrifuged and filter sterilized.
Product storage
All products have been filter sterilized and DO NOT contain antimicrobial inhibitors. Sterile technique must be used to ensure retention of Ig quality. Sodium azide may be added to a final concentration of 0.01% for long term storage at 4°C. Although we maintain our antibody supernatants at 4°C for years without loss of activity, antibody products can be frozen as aliquots by the addition of an equal volume of a cryoprotectant such as glycerol.
Supernatant Use Recommendations
Although the optimal Ig concentration for an application varies for each monoclonal antibody and must be determined empirically, a good starting concentration for immunohistochemistry (IHC) is 2-5 μg/ml. For Western blots, the concentration is decreased by one order of magnitude (that is, 0.2-0.5 μg/ml). Immunoglobulin concentration of ascites is generally >1 mg/ml, therefore a starting dilution of ascites for IHC is 1:1000
Supernatant Preparation
Frozen cells purchased from DSHB should be thawed immediately or placed into liquid nitrogen storage. To thaw cells, thaw the cells quickly in a 37°C waterbath and dispersed into 9 mls of media. Pellet the cells to remove the cryoprotectant, resuspend in 15-20 mls of media and dispense the culture into a T75 flask. Within a few days, the cells will revive and begin to proliferate. After 3 days, split the media into two T75 flasks and add 10 mls of additional media to each flask. Allow one flask to grow an additional 5 days feeding by the addition of 20 mls every 2 days. After 5 days, pellet the cell 40 ml culture, resuspend in 4 mls of media with cryoprotectant (DMSO) and freeze 4 (1ml) aliquots for long term hybridoma storage. Use the remaining T75 flask for antibody supernatant preparation. Hybridomas are not efficient in Ig secretion during log phase growth. Allow the culture to go until stationary growth is achieved. Maximum immunoglobulin concentration is reached after 10-14 days when no more dividing cells are seen. Spin out the hybridomas and discard cells, retain the supernatant and filter sterilize.
For additional information contact Karen Jensen at 319-335-3826 or dshb@uiowa.edu. | |
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