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Anti-HA (12CA5)

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Anti-HA (12CA5) For life science research only. Not for use in diagnostic procedures. Anti-HA (12CA5) Mouse monoclonal antibody (clone 12CA5) to a peptide epitope derived from the hemagglutinin protein of human influenza virus Cat. No. 11 583 816 001 200 ␮g (lyophilized) Cat. No. 11 666 606 001 5 mg (1 ml solution) y Version 07 Content version: April 2016 Store at Ϫ15 to Ϫ25°C 1. What this Product Does Purity The anti-HA monoclonal antibody is Ն 90% pure as determined by HPLC. Contents ␮ The antibody preparation does not contain pre- • Cat. No. 11 583 816 001: 200 g white lyophilizate servatives or stabilizers. • Cat. No. 11 666 606 001: 1 ml frozen liquid (5 mg/ml) 8 Affinity Ka = 1 × 10 /M Storage and Stability Isoelectric 6.4 If stored at Ϫ15 to Ϫ25°C, the lyophilized antibody preparations are Point stable until the expiration date printed on the label. For storage, prepare convenient aliquots and freeze them at Ϫ15 to 2. How to Use this Product Ϫ25°C. Avoid repeated freezing and thawing. 2.1 Before You Begin Lyophilized Anti-HA antibody is shipped at ambient temperature. Liquid Anti-HA antibody is shipped on dry ice. Epitope Tagging Before using Anti-HA to analyze the product of your target gene, Application incorporate the 27-base DNA sequence, which encodes the HA epi- tope, into the target gene sequence Anti-HA (12CA5) is used for the immunochemical detection of native using one of the following meth- influenza hemagglutinin protein and recombinant “epitope tagged” ods: proteins containing the HA epitope in western and dot blots, immuno- • Clone your gene of interest into a suitable bacterial or mammalian cytochemistry, and immunoprecipitation. expression vector. For experiments in which sensitivity is not critical, use Anti-HA • Prepare oligonucleotide linkers that can encode the HA epitope, (12CA5). For higher sensitivity detection in western blotting at 10- and clone the linkers into the target gene at the desired N-terminal, fold lower concentration use Anti-HA , High Affinity (3F10)*. C-terminal, or internal site (3). • Insert the HA-peptide coding sequence into the target gene by oli- Additional Equipment and Reagents Required gonucleotide-mediated site-directed mutagenesis (4,5). Additional equipment and reagents required to perform western blot analyses using Anti-HA (12CA5) include: Preparation of Working Solutions • Western blotting apparatus For the Iyophilized anti-HA preparation, prepare a concentrated stock • Western Transfer Buffer: 10% methanol, 24 mM Tris-base, 194 mM solution (0.4 mg/ml) by dissolving the entire Iyophilized anti-HA anti- ␮ ␮ glycine body (200 g) in 500 l PBS (rehydrate for 30 min prior to use). For storage, prepare convenient aliquots and freeze them at –15 to –25°C. • PVDF Western Blotting Membrane* The liquid anti-HA preparation (5 mg/ml) is ready to use as sup- • Washing Buffer (PBST): PBS, 0.05–2% Tween 20, pH 7.5 plied. • Blocking Solution (1:10 dilution of Western Blocking Reagent* in For most applications, dilute the Anti-HA antibody concentrate in an 1× PBST appropriate buffer to a concentration range of 0.1–5 ␮g/ml. Determine • secondary, POD-conjugated anti-mouse antibody (e.g., Goat Anti- optimal dilution buffer and dilution conditions for each specific appli- Mouse (H+L)-POD) cation and method. • Lumi-Light Western Blotting Substrate* or Lumi-LightPLUS Western Blotting Substrate* 2.2 Procedure • Lumi-Film Chemiluminescent Detection Film* Western Blotting Analysis To perform western blotting analysis using the anti-HA (12CA5), follow Product Characteristics the protocol below: Specificity Anti-HA recognizes the HA nonapeptide sequence Step Action YPYDVPDYA derived from the human influenza ³ ␮ virus hemagglutinin protein (amino acids 98-106) Perform western blot transfer to a PVDF membrane* (0.45 m (1). The antibody recognizes its antigenic determi- pore size) using Western Transfer Buffer. nant even when the HA peptide epitope is intro- The buffer must be at +2 to +8°C for electrophoretic trans- duced into unrelated recombinant proteins using a fer. technique known as “epitope tagging.” Clone 12CA5 (2) ␬ Subtype Mouse IgG2b 0416.05041112001 sigma-aldrich.com Step Action 3. Troubleshooting · Transfer the membrane (protein side up) to a container large enough to hold it. Problem Possible Cause Recommendation Be sure the side of the membrane that contains protein faces up so the detection reagents will have maximum Chemilumi- Poor isolation of Use a different cell lysis procedure access to the antigens. nescent or tagged protein chromo- A disposable, square petri dish (100 × 15 mm) makes a genic sig- Antibody too Double the concentration of the anti-HA convenient container for a 10 × 10 cm blot. nal weak or dilute and/or the secondary antibody) not visible » Too little protein Add more protein to gel. • After transfer, block the membrane for 1 hour at +15 to on the gel +25°C with 10 ml#) of Blocking Solution Poor transfer of • Increase the electrical current and/or the • Place container on a rotating platform and rotate gently for proteins from gel transfer time for the blot. 1 h at +15 to +25°C. to membrane • Be sure there are no air bubbles between the membrane and gel during transfer. Make sure the reagent completely and constantly covers the membrane during this incubation and all incubation Wrong type of For maximum signal, use PVDF membranes steps below. membrane for transfer. This incubation step may also be performed at +2 to +8°C Antibody incuba- Incubate anti-HA (and/or the secondary tion too short antibody) with the membrane blot for a lon- O/N. ger time. ¿ Drain the Blocking Solution from the container and wash the Signal develop- Double the development time. blocked membrane once with PBST. ment time too short ´ Dilute anti-HA antibody to a concentration of 0.1–1.0 ␮g/ml in Wash time too long • Shorten the washing time. a 1:20 dilution of Western Blocking Reagent in PBST. or too stringent • Omit Tween 20 from the Wash Buffer. Incubate membrane with diluted anti-HA antibody for 1 hour Enzyme on anti- Do not use sodium azide in any western blot at +15 to +25°C with gentle rotation. body conjugate reagent if you use POD-conjugated antibod- ² inactivated by ies. • Drain the antibody solution from the container and rinse the preservative membrane with approximately 20 ml#) of Wash Buffer. Substrate inactive Make fresh dilution of substrate or start with • Wash the membrane four times, 10 min per wash, with a different stock of substrate. #) 20 ml PBST. Epitope tag • Include protease inhibitors in lysis buffer. ¶ Dilute secondary, POD-conjugated antibody 1:10,000 in dilu- sequence is not • Use alternative expression system or opti- detectable due to: mize your expression system. ent (as in step 5 above) and incubate the membrane for 1 h at • Proteolytic cleav- • Insert multiple tag sequences into target +15 to +25°C with gentle rotation. age protein to increase avidity of antibody º Wash the membrane four times, 10 min per wash, with PBST. • Low level of reaction. expression • Use alternative insertion site within the tar- Prepare Lumi-Light Western Blotting Substrate* according to • Premature trans- get gene for the epitope tag sequence. package insert instructions. lation termina- ¾ tion resulting in • Drain the antibody solution from the container and rinse the loss of C-terminal membrane with approximately 20 ml#) of Wash Buffer. tag sequence • Wash the membrane four times, 10 min per wash, with High back- Antibody too con- Decrease concentration of anti-HA (and/or 20 ml#) PBST. ground, centrated Secondary Antibody) by half. additional µ Add the Lumi-Light reagent to the membrane. bands on Wash time too Wash time too short short ¸ Expose the membrane to X-ray film. For a 1 min substrate blot Incubation of Leave blot membrane in substrate for a development, perform a 1–5 min exposure initially. membrane with shorter time. The conditions for development and exposure may vary. substrate too long #) volume required per 100 cm2 membrane Wrong type mem- For minimum background, use PVDF mem- brane branes for transfer. Immunoprecipitation and immunoaffinity purification Blocking Reagent Use nonfat dry milk (5% w/v) dissolved in too dilute Reagent Diluent as Blocking Reagent. Anti-HA antibody is commonly used to isolate HA-tagged proteins and High concentrations of nonfat dry milk associated protein:protein binding partners by immunoprecipitation may reduce specific signal as well as (IP) or immunoaffinity purification. Numerous IP methods are found in background.) the literature (2, 3, 5). Optimal conditions for IP should be determined Contaminated • Use clean equipment, freshly prepared for each particular application. Use the Anti-HA Affinity Matrix (HA reagents or equip- • buffers, and new membranes. clone 3F10)* for the direct immunoprecipitation or purification of HA- ment • Always avoid touching membranes with tagged proteins. The high-affinity HA antibody is covalently coupled to bare hands; use gloves and forceps. agarose beads, which enables the rapid isolation of even rarely Secondary anti- Use an F(ab')2 fragment of a secondary anti- expressed HA-tagged protein with minimal interference of heavy- and body binds body, rather than an intact IgG. light-chain bands during western-blot analysis. untagged proteins. Heavy and light Use direct detection with peroxidase-conju- Immunofluorescence chains of primary gated monoclonal antibody to visualize Anti-HA antibody is commonly used to visualize HA-tagged fusion antibody visible on tagged proteins. proteins in various cell types by indirect immunofluorescence (6, 8-17). blot membrane For staining HA-tagged proteins in yeast, start with the method of Ber- Signal develop- Reduce development time by half. kower et al. (17). For staining of HA-tagged proteins in mammalian ment time too long cells, start with the method of Canfield and Levenson (11).
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