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Immunofluorescence analysis of mesoderm using anti-smooth muscle actin antibody (MA5-11547). Embryoid bodies (EBs) were generated from Gibco ® Human Episomal iPSC Line grown on Geltrex® in Essential 8TM Medium. After 3 weeks in culture, EBs were dissociated with TrypLETM and re-plated onto Geltrex®-coated multi-well plates. Cells were fixed, permeabilized and blocked for immunostaining. Cells were stained with a smooth muscle actin monoclonal antibody (Product # MA5-11547) at a dilution of 1:100 in 3% BSA/PBS blocking buffer overnight at 4°C, and then incubated with Alexa Fluor® 488 donkey anti mouse IgG2a antibody (Product # A21131, green) at 1:500 dilution in conjunction with NucBlue® Fixed Cell Ready Probes® Reagent. After another 3 washes, the cells were imaged on EVOS®Floid® Cell Imaging system.
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Conjugate||Alexa Fluor® 488|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against mouse IgM, mouse IgA, pooled human sera, purified human paraproteins and mouse isotypes IgG1, IgG2b and IgG3 prior to conjugation|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-10 µg/mL|
|Immunocytochemistry (ICC)||1-10 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.
|Miscellaneous PubMed (MISC)||See 12 publications below|
To minimize cross-reactivity, these goat anti-mouse IgG2a whole secondary antibodies have been affinity purified and cross-adsorbed against mouse IgM, mouse IgA, pooled human sera, purified human paraproteins, and mouse isotypes IgG1, IgG2b, and IgG3 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.
|Not Applicable||Not Cited||Restriction of receptor movement alters cellular response: physical force sensing by EphA2.||Salaita K,Nair PM,Petit RS,Neve RM,Das D,Gray JW,Groves JT||Science (New York, N.Y.) (327:1380)||2010|
|Not Applicable||Not Cited||Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue.||Robertson D,Savage K,Reis-Filho JS,Isacke CM||BMC cell biology (9:null)||2008|
|Not Applicable||Not Cited||The vascular endothelial cell mediates insulin transport into skeletal muscle.||Wang H,Liu Z,Li G,Barrett EJ||American journal of physiology. Endocrinology and metabolism (291:E323)||2006|
|Not Applicable||Not Cited||Cullin 4A-mediated proteolysis of DDB2 protein at DNA damage sites regulates in vivo lesion recognition by XPC.||El-Mahdy MA,Zhu Q,Wang QE,Wani G,Praetorius-Ibba M,Wani AA||The Journal of biological chemistry (281:13404)||2006|
|Not Applicable||Not Cited||Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis.||Kuang S,Chargé SB,Seale P,Huh M,Rudnicki MA||The Journal of cell biology (172:103)||2006|
|Not Applicable||Not Cited||Connexin 43 interacts with zona occludens-1 and -2 proteins in a cell cycle stage-specific manner.||Singh D,Solan JL,Taffet SM,Javier R,Lampe PD||The Journal of biological chemistry (280:30416)||2005|
|Not Applicable||Not Cited||Canonical transient receptor potential 1 plays a role in basic fibroblast growth factor (bFGF)/FGF receptor-1-induced Ca2+ entry and embryonic rat neural stem cell proliferation.||Fiorio Pla A,Maric D,Brazer SC,Giacobini P,Liu X,Chang YH,Ambudkar IS,Barker JL||The Journal of neuroscience : the official journal of the Society for Neuroscience (25:2687)||2005|
|Not Applicable||Not Cited||Rapid analysis of mitochondrial DNA depletion by fluorescence in situ hybridization and immunocytochemistry: potential strategies for HIV therapeutic monitoring.||Janes MS,Hanson BJ,Hill DM,Buller GM,Agnew JY,Sherwood SW,Cox WG,Yamagata K,Capaldi RA||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (52:1011)||2004|
|Not Applicable||Not Cited||Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events.||Krutzik PO,Nolan GP||Cytometry. Part A : the journal of the International Society for Analytical Cytology (55:61)||2003|
|Not Applicable||Not Cited||Chlorotoxin inhibits glioma cell invasion via matrix metalloproteinase-2.||Deshane J,Garner CC,Sontheimer H||The Journal of biological chemistry (278:4135)||2003|
|Not Applicable||Not Cited||A novel role for p120 catenin in E-cadherin function.||Ireton RC,Davis MA,van Hengel J,Mariner DJ,Barnes K,Thoreson MA,Anastasiadis PZ,Matrisian L,Bundy LM,Sealy L,Gilbert B,van Roy F,Reynolds AB||The Journal of cell biology (159:465)||2002|
|Not Applicable||Not Cited||An immunocytochemical approach to detection of mitochondrial disorders.||Hanson BJ,Capaldi RA,Marusich MF,Sherwood SW||The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (50:1281)||2002|