| Name: | Mouse Monoclonal Antibody to c-FOS, Cat# MCA-2H2 |
| Immunogen: | Full length recombinant human protein expressed in and purified from E. coli. |
| HGNC Name: | FOS |
| UniProt: | P01100 |
| Molecular Weight: | 50-65kDa |
| Host: | Mouse |
| Isotype: | IgG1 heavy, κ light |
| Species Cross-Reactivity: | Human, rat, mouse |
| RRID: | AB_2571561 |
| Format: | Purified antibody at 1mg/mL in 50% PBS, 50% glycerol plus 5mM NaN3 |
| Applications: | WB, IF/ICC, IHC |
| Recommended Dilutions: | WB: 1:500, IF/ICC or IHC: 1:500 |
| Storage: | Store at 4°C for short term, for longer term at -20°C. |
Immunofluorescent analysis of rat hippocampus section stained with mouse mAb to c-FOS, MCA-2H2, dilution 1:200, in red, and costained with rabbit pAb to FOX3/NeuN, RPCA-FOX3, dilution 1:3,000, in green. The blue is Hoechst staining of nuclear DNA. The MCA-2H2 antibody labels nuclei of spontaneously activated neurons, while FOX3/NeuN antibody stains nuclei and distal perikarya of most neurons.
Western blot analysis of cell lysates using mouse mAb to cFos, MCA-2H2, dilution 1:1,000, in green, and rabbit pAb to GAPDH, RPCA-GAPDH, dilution 1:20,000, in red, used as a loading control. [1] protein standard (red), [2] HeLa cells in serum free media. [3] HeLa cells stimulated with 20% fetal bovine serum for 2hrs after 36hrs in serum free media. [4] rat cortical neurons. [5] rat cortical neurons treated with membrane depolarization buffer for 5hrs. Multiple bands at 50-65kDa in stimulated or treated cell lysates correspond to different forms of the c-Fos proten. The single band at 37 kDa represents GAPDH protein.
Mouse Monoclonal Antibody to c-FOS
Cat# MCA-2H2
$120.00 – $800.00
The FOS gene and protein were originally identified as the transforming element in a viral oncogene. The transforming protein was named v-FOS, for viral FOS, and the normal cellular non-transforming proto-oncogene was called c-FOS, for cellular FOS. FOS is an acrynym for “FBJ murine osteogenic sarcoma”, the virus in which the gene product was first discovered. The c-FOS protein is a normal gene acting as an on/off switch controlling the expression of many other genes. The v-FOS form is mutated to stay in the on position, this persistently activating other genes and promoting unregulated cell division. The unmutated c-FOS is an “immediate-early” gene, so-called because protein expression is usually very low but increases rapidly and transiently in response to a wide array of stimuli including serum, growth factors, tumor promoters, cytokines, and UV radiation. Newly expressed c-FOS protein associates with JUN family and other basic leucine-zipper (bZIP) proteins to create a variety of activator protein-1 (AP-1) complexes (1). AP-1 complexes specifically activate the expression of many other genes and so regulate cellular responses to stimuli which may result in cell proliferation, differentiation, neoplastic transformation, apoptosis, and response to stress (2). The regulated expression of c-FOS therefore plays an important role in many cellular functions. Site specific phosphorylation activates c-FOS, while sumoylation of c-FOS inhibits the AP-1 transcriptional activity (3,4). Since c-FOS expression is induced in neurons which are rapidly firing action potentials, appropriate c-Fos antibodies can be used to identify activated neurons in tissues (5). Using current techniques it is possible to follow processes of such cells or obtain data on their mRNA expression.
The MCA-2H2 antibody was made against recombinant full length human c-FOS expressed in and purified from E. coli. It can be used to identify activated cells in cell culture and in sections and to follow c-FOS expression in western blots of cell and tissue homogenates. The antibody also works well on formalin fixed paraffin embedded sections, select the “Additional Info” for this data. The KD is 6.68 x 10-10 M, Kon rate is 1.36 x 105 1/MS and the Kdis rate is 9.12 x 10-5 1/S, all indicative of unusually high affinity. The same recombinant immunogen was used to generate a rabbit polyclonal antibody to c-FOS, RPCA-c-Fos, which has similar properties. Mouse select image at left for larger view.
As part of the characterization of this antibody we grew both HeLa cells and mixed neural cultures and compared unstimulated and stimulated cultures. Results are below. Mouse select each image for larger view.
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HeLa cells were serum-starved (Left) or serum-starved and then stimulated with 20% fetal bovine serum (FBS) for 2 hours (Right). The were then stained under identical conditions with our monoclonal antibody to c-Fos, MCA-2H2 in green, and our chicken anti-vimentin (CPCA-Vim, red). Nuclear DNA was revealed with DAPI (blue). Serum starvation inhibits c-Fos expression, while treatment with FBS for 2 hours strongly stimulates c-Fos expression which localizes in the nucleus as shown. The c-Fos antibody was used at a dilution of 1:1,000 from a 1mg/mL solution. The vimentin antibody was used at a dilution of 1:100,000. Cultures were processed using our standard fixation and staining procedure (described here). To order the c-Fos antibody go to our order form (here) or use our online store here. Picture taken with a Zeiss 40X objective and documented with a SPOT camera. Mouse click on each image to get an enlarged view.
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Rat brain neural cultures (left) and the same cells stimulated with membrane deplorization buffer for 5 hours (right). This is a salt solution containing 170mM Potassium which depolarizes and stimulates gene expression in neuronal cells but has no effect on glia. Cultures were stained with our monoclonal antibody to c-Fos, MCA-2H2 in green and rabbit anti-GFAP, RPCA-GFAP in red. Nuclear DNA is revealed in blue with the DNA stain DAPI.
Kinetic Data
We used the data shown below to derive a dissociation equilibrium constant (KD values) for this mouse monoclonal antibody. The KD is simply the ratio of the dissociation rate (koff) to the association rate (kon). Thus, KD and affinity are inversely related so that the lower the KD value, the higher the affinity of the antibody for its target. The figure below summarizes binding data for EnCor’s mouse monoclonal antibody to recombinant human c-FOS protein (MCA-2H2). MCA-2H2 displayed very strong affinity for c-FOS (KD = 0.7nM).
Above: Binding curve set for MCA-2H2 (25nM IgG) and limiting dilutions of recombinant c-FOS protein (0-350nM) obtained using our in-house label-free bio-layer interferometry system (Octet RED96). Color-coded traces show sensorgram data normalized to baseline after subtraction of 0nM IgG signal from all channels. Traces with overlying fit lines in red indicate their inclusion in the global fit analysis used to derive kinetic parameters listed under the legend (R^2 – goodness of correlation between the fit and data; kon – association rate constant; koff – dissociation rate constant; KD = koff/kon – affinity constant/equilibrium dissociation constant; see EnCor’s validation pipeline for more details). Mouse click on the image to get an enlarged view.
Chromogenic Immunohistochemistry of Formalin Fixed Paraffin Embedded Rat Material. Rat hippocampus was stained with mouse mAb to cFos, MCA-2H2, dilution 1:1,000, detected with DAB (brown) using the Vector Labs ImmPRESS method and reagents with citra buffer retrieval. Hematoxylin (blue) was used as the counterstain. MCA-2H2 specifically detects the nuclei of spontaneously active or experimentally stimulated neurons. Mouse click on the image to get an enlarged view.
Chromogenic Immunohistochemistry of Formalin Fixed Paraffin Embedded Mouse Material. Mouse hippocampus was stained with mouse mAb to cFos, MCA-2H2, dilution 1:1,000, detected in DAB (brown) following the Vector Labs Mouse on Mouse (M.O.M.®) ImmPRESS® HRP method, using the kit instructions. Hematoxylin (blue) was used as the counterstain. MCA-2H2 specifically detects the nuclei of a few spontaneously active neurons. Mouse click on the image to get an enlarged view.
1. Mildle-Langosch K. The Fos family of transcription factors and their role in tumourigenesis. Eur. J. Cancer 41:2449-2461 (2005)..
2. Chiu R, et al. The c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive genes. Cell 54:541–52 (1988).
3. Karin M. The regulation of AP-1 activity by mitogen activated protein kinases. J Biol Chem. 270:16483-6 (1995).
4. Bossis G, et al. Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation. Mol Cell Biol.25(16):6964-79 (2005).
5. Dragunow M, Faull R. The use of c-fos as a metabolic marker in neuronal pathway tracing. J. Neurosci. Mets. 29:261–265 (1989).
This is a new antibody but peer reviewed publications using it are beginning to come on line. So if you perform a Google Scholar search for “MCA-2H2” several publications will appear, or you can just select here.
