Research Technologies Branch
Flow Cytometry Section
Custom Antibodies
The NIAID Flow Cytometry Section offers NIAID investigators a cost- and time-efficient method of obtaining fluorescent labeled antibodies. Resources offered include
- Hybridoma expansion
- Antibody purification from ascites, antisera, and hybridoma supernatant
- Coupling of purified antibodies and proteins to various fluorochromes
Technology
We provide expansion of hybridoma, antibody purification, and antibody labeling. We offer consultation and advice throughout each of these processes with the goal of optimizing experiments.
Hybridoma Expansion
- Hybridoma cells supplied by the customer must be documented free of mycoplasma contamination for the safety of other cells within the facility.
- Testing for mycoplasma contamination will be performed using an outside vendor. Alternatively, certified mycoplasma-free frozen samples of the hybridoma can be sent to the facility from an accredited facility, such as ATCC.
- Two different protocols for hybridoma expansion are offered. The cells are cultured in RPMI 1640 (or a medium of the investigator's choice) in either T 650 flasks or Integra Celline CL350 flasks depending upon the anticipated yield or the investigators wishes.
- Tissue culture supernatant typically yields from 2 to 20 mg/liter depending on the cell line.
- Integra Celline CL350 typically yields 0.5 to 2.0 mg/ml.
- Supernatant can be supplied to the investigator for examination or as a final product. Our antibody production services include customer consultations at each phase of the process.
Purification
- All samples supplied by the customer must be documented free of endotoxin contamination for the safety of other samples and apparatus within the facility.
- Testing for endotoxin will be performed using an outside vendor. All apparatus used in the facility are endotoxin-free and are certified on a regular basis.
- Antibody purification is offered from ascites, antisera, and hybridoma supernatant. If the facility is expanding and purifying the hybridoma, the investigator may request a predetermined amount of purified antibody up to 100 mgs. All antibodies are supplied in sterile PBS with 0.02% sodium azide as a preservative. Other final buffers are available upon request. PAGE is routinely performed on all purified antibodies to assure the purity of the purified antibody and may be supplied to the investigator if requested. Our antibody production services include customer consultations at each phase of production.
- Ascites—Typically yields 2.0 to 10 mg/ml.
- Antisera—Purified by either Protein G affinity chromatography or preferably by affinity chromatography on an immobilized antigen-matrix. Antigen or peptide (5 – 10 mg minimum), supplied to the facility by the investigator, will be covalently attached to an activated matrix by standard techniques. This antigen-matrix may be supplied to the customer if requested.
- Hybridoma supernatant—Purified by affinity chromatography using recombinant protein G. Typical Yields: Tissue culture supernatant—2 to 20 mg/liter; Integra Celline CL250 supernatant—0.5 to 2.0 mg/ml.
- If the facility is expanding and purifying the hybridoma, the investigator may request a predetermined amount of purified antibody up to 100 mgs. All antibodies are supplied in sterile PBS with 0.02% sodium azide as a preservative. Other final buffers are available on request. PAGE is routinely performed on all purified antibodies to assure the purity of the purified antibody and may be supplied to the investigator if requested. Our antibody production services include customer consultations at each phase of production.
Labeling
- Our laboratory has more than 10 years of experience in fluorochrome modification of proteins and uses standard proven labeling protocols.
- A minimum of 0.5 mg of purified antibody is necessary for standard fluorochrome labeling.
- If enough antibody is available, we routinely test a variety of labeling procedures to determine the optimal labeling conditions of your specific antibody. A minimum of 1.5 mg of purified antibody is necessary for this conjugation optimization process.
- All antibodies are supplied in sterile PBS with 0.02% sodium azide as a preservative.
- Other final buffers are available upon request.
Custom antibodies container
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