KinomeView^® Protocol

Specific For: KinomeView® Profiling Kit #9812

NOTE: Chemiluminescence and X-ray film detection system has been validated for all the motif antibodies available in the KinomeView® kit. However, if high-resolution imaging is desired, the LI-COR Odyssey system is our recommended imaging platform.

A. SDS-PAGE:

Precast 4-20% gels routinely used for western blot analysis.

NOTE: The precast gels should be fresh (less than 4 weeks from manufacturing date). It is our experience that the quality of the results may deteriorate with precast PAGE gels that exceed 8 weeks.

B. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

• Cell Lysis Buffer (10X) #9803.
• SDS Sample Buffer (3X), Blue Loading Buffer Pack #7722, Red Loading Buffer Pack #7723.
• Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5).
• 10X Tris Buffered Saline (TBS): To prepare 1 liter of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1X).
• 10X Tris Buffered Saline with Tween 20 (TBST-10X) #9997.
• Nonfat Dry Milk #9999.
• Blocking Buffer (X-ray film): 1X TBS, 0.1% Tween-20 (TBST) with 5% w/v nonfat dry milk.
• Blocking Buffer (LI-COR): 1X TBS with 5% w/v nonfat dry milk.
• Wash Buffer: 1X TBS, 0.1% Tween-20 (TBST).
• Bovine Serum Albumin (BSA) #9998.
• Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 (TBST) with 5% BSA.
• Chemiluminescent Detection (secondary antibodies included): Phototope^®-HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody #7071; Phototope^®-HRP Western Blot Detection System, Anti-mouse IgG, HRP-linked Antibody #7072. Includes Biotinylated Protein Ladder Detection Pack #7727; Anti-rabbit IgG, HRP-linked Antibody #7074; Anti-mouse IgG, HRP-linked Antibody #7076, Anti-biotin, HRP-linked Antibody #7075, 20X LumiGLO Reagent and 20X Peroxide #7003.
• DyLight Conjugated Secondary antibodies: Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470; Anti-mouse IgG (H+L) (DyLight 800 Conjugate) #5257; Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) #5366; Anti-rabbit IgG (H+L) (DyLight 800 Conjugate) #5151.
• Prestained Protein Marker, Broad Range (Premixed Format) #7720.
• Blotting Membrane: This protocol has been optimized for nitrocellulose membranes.

C. Sample preparation and SDS-PAGE Running

1. Lyse the cell cultures or tissue samples with 1X Cell Lysis Buffer (10X) #9803, sonicate the lysate, centrifuge lysate to pellet the insoluble cellular debris.
2. Check protein concentration of the lysate. Preparing the loading stock by adjusting the protein concentration of the lysate to 1 – 2 µg/µl in SDS-PAGE loading buffer.
3. Load 20 – 30 µg protein lysate (10 – 20 µl) per lane in SDS-PAGE. If using X-ray film, also load 5 µl prestained marker and biotinylated marker mix in one of the lanes. If using LI-COR Odyssey system, also load 5 µl prestained marker in one of the lanes. Run the gel at 130 Volts until the dye reaches the bottom of the gel.
   NOTE: Biotinylated marker can be detected by anti-biotin antibody; prestained marker is autofluorescent in the LI-COR Odyssey system.
4. Electrotransfer to nitrocellulose membrane.

D. Membrane Blocking and Antibody Incubations

I. Membrane Blocking

1. Incubate membrane in protocol specific blocking buffer for one hour at room temperature with gentle agitation.
2. Rinse with TBS/T briefly.

II. Primary Antibody Incubation

1. Incubate membrane and primary antibody (1:1000) in primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 minutes each with TBS/T.

III. Secondary Antibody Incubation:

X-ray film:

1. Incubate membrane with the species appropriate HRP-conjugated secondary antibody (1:2000) and Anti-biotin, HRP-linked Antibody #7075 (1:1000) to detect biotinylated protein markers in blocking buffer with gentle agitation for one hour at room temperature.
2. Wash three times for 5 minutes each with TBS/T.
3. Proceed to detection step in section E.

LI-COR Odyssey System:

1. Incubate membrane with the species appropriate Dye-conjugated secondary antibody (1:10,000) in LI-COR blocking buffer with gentle agitation for one hour at room temperature.
2. Wash three times for 5 minutes each with TBS/T.
3. Dry the membrane at room temperature. After drying the membrane, it can then be scanned immediately or stored at room temperature to scan at a later time.

E. Detection of Proteins

X-ray film:

1. Incubate membrane with 10 ml LumiGLO (0.5 ml 20X LumiGLO, 0.5 ml 20X Peroxide, and 9.0 ml Milli-Q water) with gentle agitation for one minute at room temperature.
   NOTE: LumiGLO substrate can be further diluted if signal response is too fast.
2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and expose to x-ray film. An initial 10-second exposure should indicate the proper exposure time.
   NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following LumiGLO incubation and declines over the following 2 hours.

LI-COR Odyssey System:

1. Scan the membrane with the Odyssey Infrared Imaging System.