Immunofluorescence analysis of Chicken anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (Product # A21200) was performed using MCF-7 cells stained with Cytokeratin 19 Mouse Monoclonal Antibody (Product # MA5-12613). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with mouse primary antibody (1:250 dilution) for 3 hours at room temperature. Chicken anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate was used at concentration of 0.1 µg/ml in phosphate buffered saline containing 0.2 % BSA for 45 minutes at room temperature, for detection of cytokeratin 19 in the membrane (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI in SlowFade® Gold Antifade Mountant (Product # S36938). F-actin was stained with Rhodamine Phalloidin (Product # R415, 1:300) (Panel c: red). Panel d represents the composite image. No nonspecific staining was observed with the secondary antibody alone (panel f), or with an isotype control (panel e). The images were captured at 60X magnification.
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Chicken / IgY|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 488|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against human and rabbit IgG prior to conjugation|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:500|
|Immunocytochemistry (ICC)||0.1 µg/ml|
|Immunofluorescence (IF)||1-10 µg/mL|
|Immunohistochemistry (IHC)||1-10 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
To minimize cross-reactivity, these chicken anti-mouse IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against human and rabbit IgG prior to conjugation. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Only the nonspecific-binding secondary antibodies are captured in the column, and the highly specific secondaries flow through. The benefits of this extra step are apparent in multiplexing/multicolor-staining experiments (e.g., flow cytometry) where there is potential cross-reactivity with other primary antibodies or in tissue/cell fluorescent staining experiments where there are may be the presence of endogenous immunoglobulins.
Alexa Fluor dyes are among the most trusted fluorescent dyes available today. Invitrogen™ Alexa Fluor 488 dye is a bright, green-fluorescent dye with excitation ideally suited to the 488 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 488 dye is pH-insensitive over a wide molar range. Probes with high fluorescence quantum yield and high photostability allow detection of low-abundance biological structures with great sensitivity. Alexa Fluor 488 dye molecules can be attached to proteins at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection. The degree of labeling for each conjugate is typically 2-8 fluorophore molecules per IgG molecule; the exact degree of labeling is indicated on the certificate of analysis for each product lot.
Using conjugate solutions: Centrifuge the protein conjugate solution briefly in a microcentrifuge before use; add only the supernatant to the experiment. This step will help eliminate any protein aggregates that may have formed during storage, thereby reducing nonspecific background staining. Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. For the fluorophore-labeled antibodies a final concentration of 1-10 µg/mL should be satisfactory for most immunohistochemistry and flow cytometry applications.
We offer an extensive line of Invitrogen™ secondary antibody conjugates with well-characterized specificity and labeled with a wide selection of premium fluorescent dyes, including Invitrogen™ Alexa Fluor™ fluorescent dyes. Fluorescent secondary antibody conjugates are useful in the detection, sorting, or purification of its specified target and ideal for fluorescence microscopy and confocal laser scanning microscopy, flow cytometry, and fluorescent western detection. The breadth of fluorescent markers we offer allows our reagents to be tailored to almost any fluorescent detection system.
Secondary antibodies may be provided in three formats: whole IgG, divalent F(ab')2 fragments, and monovalent Fab fragments. Because of the high degree of conservation in the structure of many immunoglobulin domains, most class-specific secondary antibodies must be affinity-purified and cross-adsorbed to achieve minimal cross-reaction with other immunoglobulins.
Our secondary antibody conjugates are most commonly prepared by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (e.g., immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents. In the first round of purification, whole immunoglobulins binding to the immunizing antibody are recovered and mainly consist of the ~150-kDa IgG class. Further purification, for example, with Protein A or G, removes all unwanted immunoglobulin classes except the affinity-purified antibodies that react with the target-specific immunoglobulin heavy and/or light chains.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Flexibilide obtained from cultured soft coral has anti-neuroinflammatory and analgesic effects through the upregulation of spinal transforming growth factor-ß1 in neuropathic rats.
A-21200 was used in immunohistochemistry - frozen section to elucidate the role of flexibilide from cultured soft coral and its analgesic and anti-neuroinflammatory effects through upregulation of spinal transofrming growth factor-beta1 in neuropathic rat
|Chen NF,Huang SY,Lu CH,Chen CL,Feng CW,Chen CH,Hung HC,Lin YY,Sung PJ,Sung CS,Yang SN,Wang HM,Chang YC,Sheu JH,Chen WF,Wen ZH||Marine drugs (12:3792)||2014|
|Not Applicable||Not Cited||Lipoprotein lipase (LPL) is associated with neurite pathology and its levels are markedly reduced in the dentate gyrus of Alzheimer's disease brains.||Gong H,Dong W,Rostad SW,Marcovina SM,Albers JJ,Brunzell JD,Vuletic S||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (61:857)||2013|
|Not Applicable||Not Cited||Distance measurements within a concatamer of the plasma membrane Cl¿/HCO¿¿ exchanger, AE1.||Basu A,Mazor S,Casey JR||Biochemistry (49:9226)||2010|
|Not Applicable||Not Cited||Deficient ghrelin receptor-mediated signaling compromises thymic stromal cell microenvironment by accelerating thymic adiposity.||Youm YH,Yang H,Sun Y,Smith RG,Manley NR,Vandanmagsar B,Dixit VD||The Journal of biological chemistry (284:7068)||2009|
|Not Applicable||Not Cited||Filamin B mediates ICAM-1-driven leukocyte transendothelial migration.||Kanters E,van Rijssel J,Hensbergen PJ,Hondius D,Mul FP,Deelder AM,Sonnenberg A,van Buul JD,Hordijk PL||The Journal of biological chemistry (283:31830)||2008|
|Not Applicable||Not Cited||Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell.||Pucovský V,Harhun MI,Povstyan OV,Gordienko DV,Moss RF,Bolton TB||Journal of cellular and molecular medicine (11:764)||2007|
|Not Applicable||Not Cited||DNase X is a glycosylphosphatidylinositol-anchored membrane enzyme that provides a barrier to endocytosis-mediated transfer of a foreign gene.||Shiokawa D,Matsushita T,Shika Y,Shimizu M,Maeda M,Tanuma S||The Journal of biological chemistry (282:17132)||2007|
|Not Applicable||Not Cited||FAT10 plays a role in the regulation of chromosomal stability.||Ren J,Kan A,Leong SH,Ooi LL,Jeang KT,Chong SS,Kon OL,Lee CG||The Journal of biological chemistry (281:11413)||2006|
|Not Applicable||Not Cited||
Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues.
A-21200 was used in immunohistochemistry to develop three-dimensional muscle tissue architectures in vitro
|Bian W,Liau B,Badie N,Bursac N||Nature protocols (4:1522)||2009|