Page 1 of 7 INSTRUCTIONS: Z-310 Multiplex Fluorescent Western Blot Starter Kit for the Bio- Rad ChemiDoc MP Rockland Immunochemicals and Bio-Rad Laboratories have jointly developed an easy to use multiplex fluorescent western blotting protocol for use with the ChemiDoc MP system. The kit contains all of the reagents and protocols required to complete simultaneous multiplex detection of two protein targets from a complex sample on a single western blot. The procedure is fast and can be completed with the reagents contained within the kit. The resulting blot demonstrates 2-color detection of distinct protein bands labeled by a unique identifying color, allowing for the definitive identification of each target. A troubleshooting guide has also been included to adapt this procedure for use with your own samples and proteins of interest. Z-310 COMPONENT LIST (For reorder, please see reorder parts list) Reagent Description Size Blocking and dilution Blocking Buffer for Fluorescent Western Blotting 5 x 125 ml buffer Membrane Immun-Blot Low Fluorescence PVDF/Filter Paper Sets (mini) 10 sheets, 7 x 8.5 cm Membrane Immun-Blot Low Fluorescence PVDF/Filter Paper Sets (midi) 10 sheets, 8.5 x 13.5 cm Molecular Weight Precision Plus Protein WesternC Standards 25 µl Standard Control lysate HeLa, Whole cell lysate 500 µg Incubation Tray Rockland black incubation tray, large Rockland black incubation tray, small Primary antibody 1 Anti-GAPDH (RABBIT host) Polyclonal Antibody (1 µg/ µl) 50 µg Primary antibody 2 Anti α-tubulin (MOUSE host) Monoclonal Antibody (1 µg/ µl) 200 µg 12 cm x 9 cm 7.5 cm x 5 cm Secondary antibody 1 Anti-RABBIT IgG (H&L) * (GOAT) Antibody, DyLight 549 (green) 10 µg Conjugated (100 µg/ µl) Secondary antibody 2 Anti-MOUSE IgG (H&L) * (GOAT) Antibody, DyLight 649 (red) 10 µg Conjugated (100 µg/ µl) * indicates minimum cross reactivity to serum proteins form non-target species. Storage: Upon receipt store buffer at 4 C. Antibodies may be stored for up to 2 weeks at 4 C. For long -term storage, aliquot antibodies and store at -20 C. Lysate should be stored at -20 C, avoid repeated freeze/thaw cycles. Precision Plus Protein WesternC Standards should be stored at -20 C. Read all instructions before using this kit
Page 2 of 7 Important Product Information The Fluorescent Western Blotting Kit reagents are optimized to function together. For best results, use the primary and secondary antibodies at the recommended dilutions. Use a clean, dust free, incubation tray for each step of the Fluorescent Western Blotting procedure. For optimal results, agitate solutions using a rocking or rotating platform during incubation and wash steps. Always wear powder free gloves and use clean forceps to handle the blot. Please note that directly handling the blot, even with gloves, may introduce artifacts during imaging. All equipment must be clean and free of foreign material. Cover incubation trays to minimize the chance of dust or other particulate matter sticking to the membrane, as this may cause blot artifacts or an increase in background. Use pencil when marking the blot, as the fluorescent properties of pen ink may interfere with blot imaging. Additional Materials Required Criterion TGX Precast gels (4-20% or AnykD recommended), Mini-PROTEAN TGX Precast gels (AnykD recommended) or a handcast gel and electrophoresis equipment and reagents (ie.criterion Cell, Mini-PROTEAN Tetra Cell, PowerPac Power Supply)* Blotting Equipment (ie. Trans-Blot Turbo, Mini Trans-Blot or Criterion Blotter, or Trans-Blot SD Semi-Dry system)* Blotting reagents (buffers, filter paper, or Trans-Blot Turbo Transfer packs) Methanol (or ethanol) to wet the Immun-Blot LF PVDF membrane Laemmeli Sample Buffer (to dilute the WesternC Standards) Membrane wash buffer composed of Tris-buffered saline with Tween-20 (final concentration of 0.05 %) to reduce nonspecific signal Western Blot incubation tray Forceps to handle the membrane Rotary or rocking platform shaker for agitation of membrane during incubations. *This kit is compatible with standard western transfer protocols such as tank or semi-dry as well as the rapid protocols used by the Trans-Blot Turbo system. Use any suitable protocol to separate proteins by electrophoresis and transfer them to the enclosed Immun-Blot LF PVDF membrane. Protocol Overview a) Complete electrophoresis with a Criterion TGX precast gel or a Mini-PROTEAN TGX precast gel to separate the sample to be analyzed b) Transfer the gel to an Immun-Blot LF PVDF membrane using the Trans-Blot Turbo or other western blot transfer device c) Block the membrane with Rockland Blocking Buffer for Fluorescent Western Blotting d) Probe the membrane simultaneously with Rockland mouse and rabbit host primary antibodies e) Detect simultaneously with anti-mouse and anti-rabbit Rockland DyLight conjugated secondary antibodies f) Image and analyze the blot on the ChemiDoc MP
Page 3 of 7 Kit Principle This kit supplies reagents for simultaneous, 2-color, multiplex detection of two separate targets in a HeLa lysate sample. The provided antibodies for rabbit anti-gapdh and mouse anti-tubulin are be combined prior to incubation of the blotting membrane, allowing for parallel detection of these targets without the need for stripping and reprobing the membrane. Protein targets are detected using provided DyLight 549 conjugated Goat anti-rabbit antibody (green) and DyLight 649 conjugated Goat anti-mouse secondary antibody (red). Both conjugates are highly cross-absorbed to prevent cross-channel fluorescence. Additionally the kit provides Immun-Blot LF PVDF membrane, blocking and dilution buffers optimized for fluorescence blotting applications. The labeled antibodies are detected directly on the membrane using the multichannel imaging capabilities of the ChemiDoc MP system, thus eliminating film, darkrooms, and messy substrates, while preserving high sensitivity. Two-color analysis of proteins on one blot results in faster and more precise measurements of proteins. The procedures and reagents included in this kit are typical of fluorescent blotting procedures and can be adapted as needed for the Researcher s own experiments (see troubleshooting section below). A schematic illustration of the enclosed multiplex western blot is shown in figure 1. Figure 1 Schematic for 2-color multiplex western blot. After SDS-PAGE and transfer to PVDF membrane, GAPDH is detected by a rabbit anti-gapdh and the DyLight 549 (GREEN) conjugated Goat anti-rabbit secondary antibody. Tubulin is detected by a mouse anti-tubulin and the DyLight 649 (RED) Goat anti-mouse secondary antibody. Data for the immune complex is collected on the ChemiDoc MP.
Page 4 of 7 Protocol 1. Prepare buffers: - TBS: 20mM Tris-HCl, 500mM Sodium Chloride ph 7.5 - TTBS: TBS with 0.05% Tween-20 2. Sample Preparation and Gel Electrophoresis: - For the HeLa lysate, prepare by adding 10 µl 2-mercaptoethanol (final concentration of 5%). Alternatively dithiothreitol at 100 mm may be substituted for 2-mercaptoethanol. Heat sample to 95-100 C for 4-5 min. Allow sample to cool, and microfuge at maximum RPM for 1 minute. - Dilute the Precision Plus Protein WesternC Standards 1:10 (i.e. add 225 µl of Laemmeli Sample Buffer to the 25 µl sample) and load 5 µl of the diluted standard. Load 20 µl of HeLa lysate onto gel. Perform separation using a TGX Stain Free gel at 300V until the dye front reaches the J-foot (Criterion) or within 2 mm of the indicator line (Mini-PROTEAN). 3. Western Blot Transfer: - Pre-wet Immun-Blot LF PVDF membrane in 100% methanol (or ethanol) for 30 seconds then submerge the wetted membrane in water or transfer buffer for 5 min in order to remove the methanol. - Assemble the transfer apparatus according to manufacturer s instructions. For the Trans-Blot Turbo, transfer using Trans-Blot Turbo Transfer Packs, replace the included membrane with wetted Immun-Blot LF PVDF membrane. - Transfer using manufacturer s time and power recommendations for the specific gel type. - After transfer disassemble blot apparatus. Carefully recover the membrane to keep it wet*. *If the membrane shows any signs of drying, rewet it in methanol then wash in water or transfer buffer as described above. 4. Membrane Blocking: - Quickly transfer the blot into 20 ml of fluorescent western blocking buffer* - Block membrane for 1 hour with agitation at room temperature (20-25 C). *Blocking buffer should be sufficient in volume to completely cover the membrane. Use a container of the appropriate size for the membrane to ensure that reagents are not wasted. Note, if required, the membrane can be blocked overnight at 4 C 5. Primary Antibody Incubation: - Dilute both antibodies into 20 ml of blocking buffer according to the recommendations in the table below. Primary antibody Dilution Primary antibody 1 Anti-GAPDH (RABBIT host) Polyclonal Antibody 1:4000 Primary antibody 2 Anti α-tubulin (MOUSE host) Monoclonal Antibody 1:1000
Page 5 of 7 - Discard the blocking buffer from the previous step - Quickly add the primary antibody solution to the membrane (to avoid drying the membrane) and incubate with agitation for 1 hour at room temperature (or overnight at 4 C). - Discard the primary antibody solution and wash the membrane with 40mL of TTBS Wash Buffer for 5 minutes with agitation at room temperature - Repeat the wash step two additional times 6. Secondary Antibody Incubation: - Dilute both antibodies into 20 ml of blocking buffer according to the recommendations in the table below. Secondary antibody 1 Secondary antibody 2 Antibody Conjugate Anti-RABBIT IgG (H&L) * (GOAT) Antibody, DyLight 549 Conjugated Anti-MOUSE IgG (H&L) * (GOAT) Antibody, DyLight 649 Conjugated Dilution 1:2000 1:2000 - Discard the wash buffer from the previous step - Quickly add the secondary antibody solution to the membrane (to avoid drying the membrane) and incubate with agitation for 30 minutes at room temperature. Keep membrane out of direct sunlight. - Discard the secondary antibody solution and wash the membrane with 40 ml of TTBS Wash Buffer for 5 minutes with agitation at room temperature - Repeat the wash step two additional times 7. Membrane Drying: - Discard the wash solution and soak the membrane in de-ionized water for 1 minute - Transfer the membrane to 100% methanol for 30 seconds - Remove membrane using forceps and allow to fully air-dry - For extended storage, keep membrane out of direct sunlight 8. Blot Imaging Using the ChemiDoc MP: - Center blot on ChemiDoc MP UV transillluminator - Create a Multichannel Protocol. See Table 3 for application settings Table 3. Fluorescent detection settings for ChemiDoc MP Application Excitation (nm) Emission Filter (bandpass) DyLight 650 (red) 625 695 DyLight 549 (green) 530 605
Page 6 of 7 - Set Auto Image Exposure for Intense bands - Set Imaging Area to Bio-Rad Criterion Gel (for midi gels) or Bio-Rad Mini-PROTEAN Gel (for mini gels) - Use Position Gel to check the image area and adjust the position of the blot if needed - Click Run Protocol to image the blot - The blot image should look similar to Figure 2 (below) Figure 2 Expected 2-color multiplex data collected on the ChemiDoc MP. When properly performed, this protocol should provide data similar to the image shown above. Tips for Adapting Other Systems to Multiplex Fluorescent Detection: - Use primary antibodies from different host species (for example, mouse and rabbit). Antibodies produced from two closely related species such rat and mouse often demonstrate cross-reactivity with the secondary antibody, even when the antibodies are cross-adsorbed. - Use secondary antibodies that are highly cross-adsorbed against other species to avoid cross-reactivity. - Use fluorophores conjugated to secondary antibodies with distinct spectra so they can be optically distinguished from each other to avoid cross-channel fluorescence.
Page 7 of 7 - Always optimize the detection of each target singly, before simultaneous detection of multiple targets. Since some primary antibodies may be non-specific and yield multiple bands on a blot, single target detection will help determine the banding pattern of each antibody prior to a multiplex experiment. - Most membranes show higher background with shorter wavelength light. Detect your strongest target in the blue channel, your middle target in green and reserve the red channel for your weakest target. Related Products For more information about Rockland products or custom services please visit www.rockland-inc.com. For more information about these and other Bio-Rad products, visit us on the Web at discover.bio-rad.com. 변경된필드코드 DyLight is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries. This document is copyrighted. All rights are reserved. This document may not, in whole or part, be copied, photocopied, reproduced, translated, or reduced to any electronic medium or machine-readable form without prior consent, in writing, from Rockland Immunochemicals, Inc. 11/15/2011, Rockland Immunochemicals, Inc., Gilbertsville, PA, USA. All rights reserved. Z-310 REORDER PARTS LIST Reagent Description Size Part Number Blocking and dilution Blocking Buffer for Fluorescent Western Blotting 1 x 500mL MB-070 buffer Membrane Immun-Blot Low Fluorescence PVDF/Filter Paper Sets 10 sandwiches 162-0260 (mini) Membrane Immun-Blot Low Fluorescence PVDF/Filter Paper Sets 10 sandwiches 162-0262 (midi) Molecular Weight Precision Plus Protein WesternC Standards (1:10 25 µl 161-0376S Standard dilution) Control lysate HeLa, Whole cell lysate 500 µg W09-000-364 Incubation Tray Rockland black incubation tray, large Rockland black incubation tray, small 12 cm x 9 cm 7.5 cm x 5 cm WIB-4625-005 WIB-2875-010 Primary antibody 1 Anti-Glyceraldehyde-3-Phosphate Dehydrogenase 100 µg 600-401-A33 (GAPDH) (RABBIT host) Antibody Primary antibody 2 Anti α-tubulin (MOUSE host) Monoclonal Antibody 100 µg 200-301-880 Secondary antibody 1 Secondary antibody 2 Anti-RABBIT IgG (H&L) * (GOAT) Antibody, DyLight 549 (green) Conjugated Anti-MOUSE IgG (H&L) * (GOAT) Antibody, DyLight 649 (red) Conjugated 100 µg 611-142-122 100 µg 610-143-121 Rev.0 (11/15/2011)