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For a carrier free BSA and azide free version of this product see product NOTE : Please refer to primary antibody product webpage for recommended antibody dilution. Solutions and Reagents From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: Western Blotting Application Solutions Kit NOTE : Prepare solutions with reverse osmosis deionized RODI or equivalent grade water.

Dilute to 1X with dH 2 O. Nonfat Dry Milk : Wash Buffer : 1X TBST. Bovine Serum Albumin BSA : Biotinylated Protein Ladder Detection Pack : Blue Prestained Protein Marker, Broad Range kDa : Blotting Membrane and Paper : This protocol has been optimized for nitrocellulose membranes.

Pore size 0. Secondary Antibody Conjugated to HRP : Anti-rabbit IgG, HRP-linked Antibody Protein Blotting A general protocol for sample preparation. Treat cells by adding fresh media containing regulator for desired time.

Aspirate media from cultures; wash cells with 1X PBS; aspirate. Lyse cells by adding 1X SDS sample buffer µl per well of 6-well plate or µl for a 10 cm diameter plate. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube.

Keep on ice. Sonicate for 10—15 sec to complete cell lysis and shear DNA to reduce sample viscosity. Heat a 20 µl sample to 95—°C for 5 min; cool on ice. Microcentrifuge for 5 min. Load 20 µl onto SDS-PAGE gel 10 cm x 10 cm.

Electrotransfer to nitrocellulose membrane Membrane Blocking and Antibody Incubations NOTE : Volumes are for 10 cm x 10 cm cm 2 of membrane; for different sized membranes, adjust volumes accordingly.

Membrane Blocking Optional After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.

Wash three times for 5 min each with 15 ml of TBST. Primary Antibody Incubation Incubate membrane and primary antibody at the appropriate dilution and diluent as recommended in the product webpage in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.

Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody at and anti-biotin, HRP-linked Antibody at — to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.

Proceed with detection Section D. Detection of Proteins Directions for Use: Wash membrane-bound HRP antibody conjugate three times for 5 minutes in TBST. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B. Mix well. Incubate substrate with membrane for 1 minute, remove excess solution membrane remains wet , wrap in plastic and expose to X-ray film.

posted June revised June Immunoprecipitation for Native Proteins This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation. Solutions and Reagents NOTE : Prepare solutions with reverse osmosis deionized RODI or equivalent grade water.

Protein A Magnetic Beads : Magnetic Separation Rack : or ATP 10 mM for kinase assays : To prepare 0. Preparing Cell Lysates Aspirate media. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.

Remove PBS and add 0. Scrape cells off the plate and transfer to microcentrifuge tubes. Sonicate on ice three times for 5 sec each. Microcentrifuge for 10 min at 4°C, 14, x g and transfer the supernatant to a new tube. The supernatant is the cell lysate.

If necessary, lysate can be stored at °C. Immunoprecipitation Cell Lysate Pre-Clearing Optional A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads.

Briefly vortex the stock tube to resuspend the magnetic beads. IMPORTANT : Pre-wash magnetic beads just prior to use: Transfer 20 μl of bead slurry to a clean tube.

Place the tube in a magnetic separation rack for seconds. Add μl cell lysate to 20 μl of pre-washed magnetic beads. Incubate with rotation for 20 minutes at room temperature. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.

Proceed to immunoprecipitation section. Immunoprecipitation IMPORTANT : Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation.

Add primary antibody at the appropriate dilution as recommended in the product datasheet to µl cell lysate. Incubate with rotation overnight at 4°C. to form the immunocomplex. Pre-wash magnetic beads see Cell Lysate Pre-Clearing section, steps 1 and 2.

Transfer the lysate and antibody immunocomplex solution to the tube containing the pre-washed magnetic bead pellet. Incubate with rotation for 20 min at room temperature. Pellet beads using magnetic separation rack. Wash pellets five times with μl of 1X cell lysis buffer.

Keep on ice between washes. Proceed to analyze by western immunoblotting or kinase activity section D. Sample Analysis Proceed to one of the following specific set of steps. For Analysis by Western Immunoblotting Resuspend the pellet with µl 3X SDS sample buffer, briefly vortex to mix, and briefly microcentrifuge to pellet the sample.

Heat the sample to °C for 5 min. Transfer the supernatant to a new tube. The supernatant is the sample. Analyze sample by western blot see Western Immunoblotting Protocol. For Analysis by Kinase Assay Wash pellet twice with µl 1X kinase buffer.

Suspend pellet in 40 µl 1X kinase buffer supplemented with µM ATP and appropriate substrate. Incubate for 30 min at 30°C. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.

Transfer supernatant containing phosphorylated substrate to another tube. Heat the sample to °C for min and microcentrifuge for 1 min at 14, x g. Load the sample µl on SDS-PAGE gel. posted December revised April Immunohistochemistry Paraffin A. Deionized water dH 2 O.

Hematoxylin optional. Wash Buffer : 1X Tris Buffered Saline with Tween ® 20 TBST : To prepare 1L 1X TBST add ml 10X Tris Buffered Saline with Tween ® 20 to ml dH 2 0, mix.

SignalStain ® Antibody Diluent Detection System : SignalStain ® Boost IHC Detection Reagents HRP, Rabbit Substrate : SignalStain ® DAB Substrate Kit Hematoxylin: Hematoxylin Mounting Medium: SignalStain ® Mounting Medium Wash sections two times in dH 2 O for 5 min each.

Antigen Unmasking For Citrate : Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature 95°°C.

Staining Wash sections in dH 2 O three times for 5 min each. Wash sections in dH 2 O two times for 5 min each. Wash sections in wash buffer for 5 min. Block each section with — µl of preferred blocking solution for 1 hr at room temperature. Remove blocking solution and add — µl primary antibody diluted in SignalStain ® Antibody Diluent to each section.

Incubate overnight at 4°C. Equilibrate SignalStain ® Boost Detection Reagent HRP, Rabbit to room temperature. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

Cover section with 1—3 drops SignalStain ® Boost Detection Reagent HRP, Rabbit as needed. Incubate in a humidified chamber for 30 min at room temperature. Wash sections three times with wash buffer for 5 min each.

Add 1 drop 30 µl SignalStain ® DAB Chromogen Concentrate to 1 ml SignalStain ® DAB Diluent and mix well before use. Apply — µl SignalStain ® DAB to each section and monitor closely. Immerse slides in dH 2 O.

If desired, counterstain sections with hematoxylin Repeat in xylene, incubating sections two times for 10 sec each. Mount sections with coverslips and mounting medium Immunofluorescence Immunocytochemistry A. Solutions and Reagents NOTE: Prepare solutions with reverse osmosis deionized RODI or equivalently purified water.

Adjust pH to 8. cat , use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use. Mix well then add 0. Specimen Preparation - Cultured Cell Lines IF-IC NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips. NOTE: Formaldehyde is toxic, use only in fume hood.

Allow cells to fix for 15 minutes at room temperature. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each. Proceed with Immunostaining Section C.

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