MO22125
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Data Sheet (ACTA1, ACTA2, ACTC1, ACTB, ACTG1 and ACTG2) Catalog Number: Product Type: Immunogen Sequence: Mouse Monoclonal Species Reactivity: Actin preparation derived from bovine brain. Mouse Human, Rat, Mouse, Bovine, Porcine* 100 ul. Liquid at a concentration of 1 mg/ml. Immunofluorescence: 1:500-1:1,000 Western Blot: 1:1,000-1:5,000 (major band at 42kDa) Dilutions listed as a recommendation. Optimal dilution should be determined by investigator. Antibody can also be aliquotted and stored frozen at -20° C to -70° C in a manual defrost freezer for six months without detectable loss of activity. The antibody can be stored at 2° - 8° C for 1 month without detectable loss of activity. Avoid repeated freeze-thaw cycles. Application Notes. *Also works on 3T3, Hek293, HeLa and other common cell types grown in tissue culture. This antibody binds to all six isotypes of mammalian actin. Immunostaining Cell Cultures 1. Draw of culture medium with aspirator and add 1 ml of 3.7 % formalin in PBS solution to the dish. (make up from 10mls Fisher 37% formalin plus 90mls PBS, the Fisher formalin contains 37% formaldehyde plus about 1% methanol which may be relevant sometimes). Let sit at room temp for 1 minute. (can add 0.1% Tween 20 to PBS used here and all subsequent steps to reduce background; probably best not to do this first time round though as it may extract your antigen or help wash your cells off the dish). 2. Take off the formalin/PBS and add 1ml of cold methanol (-20°C, kept in well sealed bottle in fridge). Let sit for no more than 1 minute. 3. Take off methanol and add 1ml of PBS, not letting the specimen dry out. To block nonspecific antibody binding can add ~10ml (=1%) of goat serum (Sigma), and can incubate for 30 minutes. Can then add antibody reagents. Typically 100ml of hybridoma tissue culture supernatent or 1ml of mouse ascites fluid or crude serum. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight, exact time not too critical). Can do very gentle shaking for well adherent cell lines (3T3, Hek293 etc.). 4. Remove primary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS. 5. Add 0.5 mls of secondary antibody. These are fluorescently labeled Goat anti mouse antibodies and are conjugated to ALEXA dyes and are from Molecular probes (Eugene Oregon, the ALEXA dyes are sulphonated rhodamine compounds and are much more stable to UV than FITC, TRITC, Texas red etc.). Typically make 1:2,000 dilutions of these secondaries in PBS plus 1% goat serum, BSA or non fat milk carrier. Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour, or can do 4°C overnight). Can do gentle shaking for well adherent cell lines (3T3, HEK293 etc.). 6. Remove secondary antibody and replace with 1 ml of PBS. Let sit for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS. 7. Drop on one drop of Fisher mounting medium onto dish and apply 22mm square c overslip. View in the microscope! Immunostaining Tissue FOR RESEARCH USE ONLY NEUROMICS’ REAGENTS ARE FOR IN VITRO AND CERTAIN NON-HUMAN IN VIVO EXPERIMENTAL USE ONLY AND NOT INTENDED FOR USE IN ANY HUMAN CLINICAL INVESTIGATION, DIAGNOSIS, PROGNOSIS, OR TREATMENT. THE ABOVE ANALYSES ARE MERELY TYPICAL GUIDES. THEY ARE NOT TO BE CONSTRUED AS BEING SPECIFICATIONS. ALL OF THE ABOVE INFORMATION IS, TO THE BEST OF OUR KNOWLEDGE, TRUE AND ACCURATE. HOWEVER, SINCE THE CONDITIONS OF USE ARE BEYOND OUR CONTROL, ALL RECOMMENDATIONS OR SUGGESTIONS ARE MADE WITHOUT GUARANTEE, EXPRESS OR IMPLIED, ON OUR PART. WE DISCLAIM ALL LIABILITY IN CONNECTION WITH THE USE OF THE INFORMATION CONTAINED HEREIN OR OTHERWISE, AND ALL SUCH RISKS ARE ASSUMED BY THE USER. WE FURTHER EXPRESSLY DISCLAIM ALL WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. V1-05/2012 www.neuromics.com Neuromics Antibodies • 5325 West 74th Street, Suite 8 • Edina, MN 55439 phone 866-350-1500 • fax 612-677-3976 • e-mail pshust

Solutions PBS - sodium phosphate-buffered (100 mM; pH 7.2) isotonic (0.9% NaCl, w/v) saline Antibody dilution buffer (PBS with 0.1% non-ionic detergent, such as Triton X-100 or Tween-20) fluorescein anti-fading reagent -- Make up a 2 mg/ml phenylene diamine solution in PBS (phenylene diamine requires extensive vortexing to put it into solution). Once the phenylene diamine is completely dissolved, add an equal volume of glycerol and mix. This reagent will last about a week at O 20 C. Discard this reagent when it starts to turn dark brown. Other Reagents Fluorescein-labeled goat anti-mouse...

4. Wash membranes in TBS (10mM Tris, 154mM NaCl, pH=7.5 plus 0.1% Tween 20) for 3 times at least five minutes each time with extensive agitation. A. Alkaline Phosphatase Blot System 1. Incubate in alkaline phosphatase conjugated antibody against the primary antibody (e.g. Goat anti-mouse, rabbit or chicken; buy from Sigma or some other trusted source). Typical concentration is 1:1,000 in TBS (10mM Tris/HCl, 154mM NaCl, pH=7.5). Add a small amount of BSA or NFM to act as carrier. Incubate for 1 hour at room temperature (or 37°C) with shaking. 2. Wash in TBS three times 5 minutes each. (N.B....

Image: HeLa cells stained with Actin (red) and also with our chicken polyclonal antibody to Vimentin, (green). The actin antibody stains the submembranous actin rich cytoskeleton and also stress fibers, bundles of actin associated with adhesion sites. The vimentin antibody stains a quite different cytoskeletal network, the intermediate filaments. The blue stain reveals DNA in the nuclei of these cells. Image: Western blot of crude extract of the human carcinoma HeLa cell line. Lane 15 was probed with Actin, our monoclonal antibody to all six actin isotypes, giving a strong clean band at an...