secondary antibodies
Affinity purified anti-IgG (H+L) conjugates
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Immunodetection |
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secondary antibodies Affinity purified anti-IgG (H+L) conjugates |
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Secondary antibodies from Uptima are high quality, cost-effective antibodies, which can be used for a wide variety of immunological applications. |
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| Technical Information |
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All Uptima antibodies are available un-conjugated or covalently coupled to a variety of labels (e.g. biotin, peroxidase, alkaline phosphatase, fluorescein, etc). |
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Label |
Unlabeled |
Biotin |
FITC |
Phycoerythrine |
Peroxidase |
Alkaline |
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Phosphatase |
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Buffer |
PBS (a) |
PBS (b) |
PBS (b) |
PBS (e) |
PBS (c) |
TBS (d) |
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IgG (whole) |
4mg ; 1 ml |
1mg ; 1ml |
1mg ; 1ml |
100 tests ; 1ml |
1mg ; 1ml |
1mg ; 1ml |
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4mg / ml |
1mg/ml |
1mg/ml |
- |
1mg/ml |
1mg/ml |
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F(ab')2 |
2mg ; 1ml |
0.5mg ; 0.5ml |
0.5mg ; 0.5ml |
100 tests ; 1m |
0.5mg ; 0.5ml |
0.5mg ; 0.5ml |
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2mg / ml |
1mg/ml 1 |
1mg/ml |
- |
1mg/ml |
1mg/ml |
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Storage |
- 20°C |
- 20°C or +4°C |
+4°C |
+4°C |
+4°C |
+4°C |
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>2 years |
2 years |
2 years |
6 months |
1 year |
1 year |
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Dilution of use |
Depends on |
1/1000-1/20000 |
1/50-1/200 |
1/10 - 1/100 |
1/1000 - 1/16000 |
1/1000 - 1/16000 |
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Technique |
(ELISA) |
(CMF) |
(CMF) |
(ELISA) |
(ELISA) |
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Applications |
ELISA - IHC |
ELISA - IH |
FCM - IF |
FCM |
ELISA - IH |
ELISA - IH |
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WB - FCM -IF controls-latex- Aggl.-Oucht.-IEF Immunocapture T... |
WB - FCM - IF |
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WB |
WB |
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(a) PBS (NaCl 150mM, phosphate 10mM, pH7,4), NaN3 0,09%, Glycerol 20% |
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(b) PBS (NaCl 150mM, phosphate 10mM, pH7,4) + BSA 0,2%, NaN3 0,09%, Glycerol 20% |
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(c) PBS (NaCl 150mM, phosphate 10mM, pH7,4) + BSA 0,2%, Thimerosal 0,01%, Glycerol 20% |
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(d) TBS (NaCl 150mM, Tris 50mM, pH8,0, 1mM MgCl2) + BSA 0,2%, 0,09% NaN3, Glycerol 20% |
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(e) PBS (NaCl 150mM, phosphate 10mM, pH7,4) + BSA 0,2%, NaN3 0,09% |
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Unlabeled affinity purified IgGs Unlabeled antibodies are typically coated onto ELISA plates or latex particles to capture antigens or antigen specific antibodies, which allows for better orientation ( see also our coated plates). They are also used in immunodetection systems by precipitation onto gels (Ouchterlony, Manciny, IEF...), in indirect agglutinations, in immunocapture techniques to dilute antibodies with a too high affinity to saturate immunoglobulin receptors on cells and to prevent unspecific binding of a labeled antibody from the same species.Abs specific activity is checked by ELISA with a coated IgG, similar to the one used in the immunization and purification. Purity is checked by electrophoresis. |
Pre-adsorbed affinity purified antibodies Secondary antibodies may cross-react with serum proteins from other species, although affinity purified, beside the reaction with the original IgG used for immunization and purification. For some applications this cross-reactivity is unwanted and a more specific antibody is required. The cross-reactivity is reduced by removing the undesired cross-reactive antibodies during a pre-adsorbtion step on an affinity column, which has been loaded with serum proteins from selected species. The specificity of this pre-adsorbed antibody is then checked against IgG’s from different species in an ELISA to guarantee that the pre-adsorbed antibody has minimum cross-reactivity with the stated species. This selectivity is indicated by a short annotation following the name of the antibody : i.e. Min x Rt indicates a minimal binding of the relevant antibody to rat serum proteins. |
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Affinity purified F(ab’)2 F(ab’)2 fragments are prepared by enzymatic cleavage of IgG’s followed by a purification step to remove the Fc fragments. Resulting F(ab’)2 remain bivalent and will bind antigens with the same affinity as whole IgGs. F(ab’)2 fragments are generally recommended for immunoassays involving cells (lymphocytes, macrophages, etc). Whole IgGs may produce high backgrounds in these assays, as their Fc fragments may be recognized by immunoglobulin receptors. The use of F(ab’)2 fragments will avoid such undesired binding, increasing the specificity and sensitivity of the assay without altering the affinity of the antibody. |
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FITC affinity purified antibodies Fluorescein (FITC) is a commonly used fluorescent label with an excitation wavelength of 495 nm (argon laser), and an emission at 528 nm. Uptima FITC labeled antibodies are conjugated with 4-8 fluorophores per molecule to achieve the best signal to noise ratio. |
PE affinity purified antibodies B-phycoerythrin (PE), isolated from Porphyridium cruentum, is a red fluorescent dye (optimum excitation at 495 nm (from Argon (488 nm) or Mercury (545 nm) lamps), emission at 595 nm (high quantum of 0.68)). B-PE is better excited at 545 nm and gives a more stable fluorescent signal, when compared with R-PE. B-phycoerythrin and FITC can be used together for double analysis in FCM, as both these fluorophores are excited with the same lamps, but they emit at distinctively different wavelengths. |
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Enzymatic Labels - Selection Guide |
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HRP affinity purified antibodies Uptima antibodies are available labeled with horseradish peroxidase (HRP). The peroxidase is selected for its high activity and conjugated to the antibodies following an optimized process, which results in highly sensitive and stable antibodies. Peroxidase is one of the most commonly used enzymes as it is cheap and versatile, with an extensive range of soluble and insoluble substrates available. Recommended colorimetric substrates for HRP are TMB for ELISA (cat. # UP66478, UPS0817, UPS0818), DAB (Cat.# 67992) and TMB for blotting (cat.# UP15426). Higher sensitivity can be achieved by using a chemiluminescent substrate (UP99619). One of the primary problems associated with HRP is non-specific staining that results form endogenous peroxidase activity within immunocytochemistry applications. |
AP affinity purified antibodies Alkaline Phosphatase (AP) is an enzyme, which is isolated from calf intestines. It gives a more linear activity than peroxidase, and is suitable for most immunodetections. Alkaline phosphatase is especially recommended for applications, where high levels of endogenous peroxidase are present. Because reaction rates remain linear when using AP, the sensitivity can be increased by just allowing the reaction to proceed for longer periods of time. Recommended substrates for alkaline phosphatase are : BCIP/NBT for blotting and IHC applications (cat.# UP09605) and pNPP for ELISA (cat.# UP73250). Endogenous alkaline phosphatase activity found in some samples can be inhibited by levamisole. The reaction with pNPP allows kinetic readings. |
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Biotin has a very high affinity for avidin and related molecules (neutralized avidin, streptavidin Ka=10-14 mol-1 ). Biotin is a very popular and versatile label, used with streptavidin conjugates and other amplifying systems. Uptima biotinylated antibodies carry 1–3 biotins per IgG or F(ab’) 2 molecule.They are recommended for use with streptavidin-FITC (UP277130), streptavidin-HRP (UP395880), Streptavidin-AP (UP518490), Streptavidin-PE (UP777760). |
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