Western blot analysis was performed on whole cell extracts (30 µg lysate) of K-562 (Lane 1) and U-87 MG (Lane 2). The blots were probed with Anti-SOD2 Mouse Monoclonal Antibody (Product # MA1-106, 2 µg/ml) and detected using Rabbit anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 680 (Product # A21057) at dilutions 0.2 µg/ml (Fig. 1), 0.1 µg/ml (Fig. 2) and 0.04 µg/ml (Fig. 3). A 22 kDa band corresponding to SOD2 was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary antibody after blocking with 5 % skimmed milk. Fluorescent detection was performed using the Odyssey® Fc imaging system (Li-cor Biosciences).
|Tested species reactivity||Mouse|
|Published species reactivity||Not Applicable|
|Host / Isotype||Goat / IgG|
|Immunogen||Gamma Immunoglobins Heavy and Light chains|
|Conjugate||Alexa Fluor® 680|
|Storage buffer||PBS, pH 7.5|
|Contains||5mM sodium azide|
|Storage Conditions||4° C, store in dark|
|Cross Adsorption||Against human IgG and human serum prior to conjugation|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:250-1:500|
|Immunofluorescence (IF)||1-10 µg/mL|
|Western Blot (WB)||0.04-0.2 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 goat anti-mouse IgG (H+L) whole secondary antibodies have been affinity purified and cross-adsorbed against human IgG and human serum 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 680 dye is a bright, near-infrared-fluorescent dye with excitation ideally suited to the 680 nm laser line. For stable signal generation in imaging and flow cytometry, Alexa Fluor 680 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 680 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.
Age-related attenuation of parasympathetic control of the heart in mice.
A-21057 was used in western blot to propose that decreased acetylcholine synthesis or release and alterations in the choline transporter contribute to parasympathetic nervous system dysfunction
|Freeling JL,Li Y||International journal of physiology, pathophysiology and pharmacology (7:126)||2016|
Release of tensile strain on engineered human tendon tissue disturbs cell adhesions, changes matrix architecture, and induces an inflammatory phenotype.
A-21057 was used in western blot to characterize engineered human tendon tissue and how release of tensile strain changes matrix architecture, disturbs cell adhesions, and induces an inflammatory phenotype
|Bayer ML,Schjerling P,Herchenhan A,Zeltz C,Heinemeier KM,Christensen L,Krogsgaard M,Gullberg D,Kjaer M||PloS one (9:null)||2014|
|Not Applicable||Not Cited||Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria.||Elustondo PA,White AE,Hughes ME,Brebner K,Pavlov E,Kane DA||The Journal of biological chemistry (288:25309)||2013|
|Not Applicable||Not Cited||Nucleotide excision repair-induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response.||Marteijn JA,Bekker-Jensen S,Mailand N,Lans H,Schwertman P,Gourdin AM,Dantuma NP,Lukas J,Vermeulen W||The Journal of cell biology (186:835)||2009|
|Not Applicable||Not Cited||Fibroblast growth factor receptor 1 is a key regulator of early adipogenic events in human preadipocytes.||Widberg CH,Newell FS,Bachmann AW,Ramnoruth SN,Spelta MC,Whitehead JP,Hutley LJ,Prins JB||American journal of physiology. Endocrinology and metabolism (296:E121)||2009|
|Not Applicable||Not Cited||alpha-Actinin interacts with rapsyn in agrin-stimulated AChR clustering.||Dobbins GC,Luo S,Yang Z,Xiong WC,Mei L||Molecular brain (1:null)||2008|
|Not Applicable||Not Cited||Quantitative evaluation of signaling events in Drosophila S2 cells.||Bond D,Primrose DA,Foley E||Biological procedures online (10:20)||2008|
|Not Applicable||Not Cited||Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix.||Bass MD,Roach KA,Morgan MR,Mostafavi-Pour Z,Schoen T,Muramatsu T,Mayer U,Ballestrem C,Spatz JP,Humphries MJ||The Journal of cell biology (177:527)||2007|
|Not Applicable||Not Cited||Tumor necrosis factor receptor 2 signaling induces selective c-IAP1-dependent ASK1 ubiquitination and terminates mitogen-activated protein kinase signaling.||Zhao Y,Conze DB,Hanover JA,Ashwell JD||The Journal of biological chemistry (282:7777)||2007|
|Not Applicable||Not Cited||Overexpression of Na(+)/K (+)-ATPase parallels the increase in sodium transport and potassium recycling in an in vitro model of proximal tubule cellular ageing.||Silva E,Gomes P,Soares-da-Silva P||The Journal of membrane biology (212:163)||2007|
|Not Applicable||Not Cited||Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.||Donkor J,Sariahmetoglu M,Dewald J,Brindley DN,Reue K||The Journal of biological chemistry (282:3450)||2007|
|Not Applicable||Not Cited||Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1.||Aracena-Parks P,Goonasekera SA,Gilman CP,Dirksen RT,Hidalgo C,Hamilton SL||The Journal of biological chemistry (281:40354)||2006|
|Not Applicable||Not Cited||Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2.||Zehe C,Engling A,Wegehingel S,Schäfer T,Nickel W||Proceedings of the National Academy of Sciences of the United States of America (103:15479)||2006|
|Not Applicable||Not Cited||Protein phosphatase 6 subunit with conserved Sit4-associated protein domain targets IkappaBepsilon.||Stefansson B,Brautigan DL||The Journal of biological chemistry (281:22624)||2006|
|Not Applicable||Not Cited||Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX.||Zhu M,Rhee I,Liu Y,Zhang W||The Journal of biological chemistry (281:18408)||2006|
|Not Applicable||Not Cited||A novel cell-based assay for G-protein-coupled receptor-mediated cyclic adenosine monophosphate response element binding protein phosphorylation.||Selkirk JV,Nottebaum LM,Ford IC,Santos M,Malany S,Foster AC,Lechner SM||Journal of biomolecular screening (11:351)||2006|
|Not Applicable||Not Cited||TLR1- and TLR6-independent recognition of bacterial lipopeptides.||Buwitt-Beckmann U,Heine H,Wiesmüller KH,Jung G,Brock R,Akira S,Ulmer AJ||The Journal of biological chemistry (281:9049)||2006|
|Not Applicable||Not Cited||Neutral ceramidase encoded by the Asah2 gene is essential for the intestinal degradation of sphingolipids.||Kono M,Dreier JL,Ellis JM,Allende ML,Kalkofen DN,Sanders KM,Bielawski J,Bielawska A,Hannun YA,Proia RL||The Journal of biological chemistry (281:7324)||2006|
|Not Applicable||Not Cited||Pharmacological promotion of inclusion formation: a therapeutic approach for Huntington's and Parkinson's diseases.||Bodner RA,Outeiro TF,Altmann S,Maxwell MM,Cho SH,Hyman BT,McLean PJ,Young AB,Housman DE,Kazantsev AG||Proceedings of the National Academy of Sciences of the United States of America (103:4246)||2006|
|Not Applicable||Not Cited||Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II.||Foulquier F,Vasile E,Schollen E,Callewaert N,Raemaekers T,Quelhas D,Jaeken J,Mills P,Winchester B,Krieger M,Annaert W,Matthijs G||Proceedings of the National Academy of Sciences of the United States of America (103:3764)||2006|
|Not Applicable||Not Cited||Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging.||Yasuda R,Harvey CD,Zhong H,Sobczyk A,van Aelst L,Svoboda K||Nature neuroscience (9:283)||2006|
|Not Applicable||Not Cited||The glycosphingolipid globotriaosylceramide in the metastatic transformation of colon cancer.||Kovbasnjuk O,Mourtazina R,Baibakov B,Wang T,Elowsky C,Choti MA,Kane A,Donowitz M||Proceedings of the National Academy of Sciences of the United States of America (102:19087)||2005|
|Not Applicable||Not Cited||The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin.||Qin Y,Capaldo C,Gumbiner BM,Macara IG||The Journal of cell biology (171:1061)||2005|
|Not Applicable||Not Cited||The membrane-bound histidine acid phosphatase TbMBAP1 is essential for endocytosis and membrane recycling in Trypanosoma brucei.||Engstler M,Weise F,Bopp K,Grünfelder CG,Günzel M,Heddergott N,Overath P||Journal of cell science (118:2105)||2005|
|Not Applicable||Not Cited||Visualization and quantitation of peroxisomes using fluorescent nanocrystals: treatment of rats and monkeys with fibrates and detection in the liver.||Colton HM,Falls JG,Ni H,Kwanyuen P,Creech D,McNeil E,Casey WM,Hamilton G,Cariello NF||Toxicological sciences : an official journal of the Society of Toxicology (80:183)||2004|
|Not Applicable||Not Cited||Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.||Matheny SA,Chen C,Kortum RL,Razidlo GL,Lewis RE,White MA||Nature (427:256)||2004|