Sep 28, 2018
Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit–IRDye® 680RD
- LI-COR Biosciences1
- 1LI-COR Biosciences
- LI-COR Biosciences
External link: https://www.licor.com/documents/obwvw3w5ku7vmmezmqbl
Protocol Citation: LI-COR Biosciences 2018. Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit–IRDye® 680RD. protocols.io https://dx.doi.org/10.17504/protocols.io.gvkbw4w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 29, 2016
Last Modified: September 28, 2018
Protocol Integer ID: 4748
Abstract
Immunoprecipitation (IP) is a valuable tool that utilizes antibody: antigen interactions to isolate a specific target protein from thousands of proteins in a complex solution. The specificity of the reaction increases the concentration of dilute proteins as high as 10,000-fold; a property that renders the technique useful in the examination of rare proteins. With careful choice of antibodies, a co-IP can also be used to demonstrate protein interaction partners.
Attachments
Guidelines
I. Introduction
Immunoprecipitation (IP) is a valuable tool that utilizes antibody: antigen interactions to isolate a specific target protein from thousands of proteins in a complex solution. The specificity of the reaction increases the concentration of dilute proteins as high as 10,000-fold; a property that renders the technique useful in the examination of rare proteins. With careful choice of antibodies, a co-IP can also be used to demonstrate protein interaction partners*.
The typical workflow for an IP can be divided into four parts:
Incubation of the primary antibody with the sample (e.g., a cell lysate);
Capture of the antibody: antigen complex using a solid support such as Protein A agarose beads;
Separation of the proteins by SDS-PAGE;
Detection of the target protein by Western blot.
The technique is time-consuming and often requires optimization of antibody and lysate concentrations as well as the inclusion of appropriate positive and negative controls. Ultimately, success relies on the choice of antibodies, not only for the IP step but also for Western blot detection of post-IP proteins. A common solution to avoid detection of the heavy and light chains from the IP step in the Western blot is to use two separate antibodies to the same target but from different host species. An example would be using a mouse capture antibody for IP and a rabbit antibody for post-IP Western blot detection. The LI-COR Quick Western Kit-IRDye 680RD eliminates the need for two separate antibodies in the IP workflow. The proprietary Detection Reagent does not recognize denatured heavy or light chains from monoclonal antibodies; therefore, the same monoclonal antibody can be used for the IP step AND post-IP Western blot detection. In addition, the Quick Western Protocol saves 90 minutes of assay time compared to a traditional two-step Western blot protocol*.
*NOTE: The data, procedure, and troubleshooting outlined in this Technical Note are provided as guidelines. A complete discussion of the IP technique and the Western blot technique are beyond the scope of this material.
The following data simulate post-IP Western blot detection utilizing traditional IRDye secondary antibody detection (panel A) compared with Quick Western Kit-IRDye 680RD detection (panel B). The traditional detection method recognizes the denatured heavy and/or light chains of the IP antibodies. The Quick Western Kit detection method does not recognize any denatured heavy and light chains from the IP antibodies evaluated. This lack of recognition is the foundation for single antibody IP and post-IP Western blot detection.
NOTE: The Detection Reagent will recognize denatured heavy or light chains of certain polyclonal antibodies. It will not recognize denatured heavy or light chains of monoclonal (mouse or rabbit) antibodies.
II. Required Reagents
Quick Western Kit-IRDye 680RD (LI-COR, P/N 926-68100)
A431 Cell Lysate in 50 mM Tris-HCl pH7.8, 150 mM NaCl, 1% NP-40, 0.2% Sodium Deoxycholate, 1X Halt Protease Inhibitor
Protein A Beads (Thermo Fisher, P/N 20333)
Anti-ERK2 Mouse Antibody (Santa Cruz Biotechnology, P/N SC1647)
1XPBS
Tween® 20
4X Protein Sample Loading Buffer (LI-COR, P/N 928-40004). Dilute to 2X with water.
26.5-gauge needle
3 mL syringe
20X MES Buffer (Invitrogen, P/N NP0002)
NuPAGE® Antioxidant (Invitrogen P/N NP0005)
NuPAGE 10% Bis-Tris gels (Invitrogen P/N NP0303BOX)
Odyssey® Protein Molecular Weight Marker (LI-COR® P/N 928-40000)
Tris-Glycine Buffer
Nitrocellulose (LI-COR P/N 926-31092)
Odyssey Blocking Buffer (PBS) (LI-COR P/N 927-40000)
IRDye 680RD Goat anti-Mouse (LI-COR P/N 926-68070)
III. Sample Protocol: Immunoprecipitation and Detection of ERK2
NOTE: This is intended as a general guideline for an IP and Western blot procedure. Different targets will require optimization of antibody concentrations, lysate concentrations, and incubation times. In addition, solid supports other than Protein A agarose beads may be substituted.
IMPORTANT: It is critical that the antibody chosen is recommended for both immunoprecipitation AND Western blot by the primary antibody vendor. This information can be found in the primary antibody pack insert or on the vendor’s website.
WARNING: Mouse IgG1 subclass antibodies may not work in this procedure, or may require additional optimization.
See 'STEPS' for protocol.
IV. Experimental Results
V. Experimental Considerations
Negative and positive controls are extremely important for correct interpretation of the data and should ALWAYS be included in an immunoprecipitation experiment.
Careful consideration should be given in the choice of solid support. Be sure that your selection provides the best binding affinity to the type of antibody used. Protein A-sepharose, agarose, magnetic beads, etc., are best for mouse IgG2a, IgG2b, or rabbit IgG. Protein G has a higher affinity for mouse IgG1. Consult the manufacturer’s guide if you are uncertain of the best support for
your experiment.
Mouse or rabbit monoclonal antibodies are recommended for use with this kit (for IP and detection of post-IP proteins by Western blot). The detection reagent recognizes denatured heavy and/or light chains of certain polyclonal antibodies, which may interfere with post-immunoprecipitation detection by Western blot.
If there are cross-reacting bands in the lysate + PBS (negative control) as well as your lysate + antibody lanes, pre-clearing the lysates is an option to reduce non-specific contaminants and remove proteins with high affinity to Protein A or G.
– Wash an amount of your solid support equal to 30 μL of 50:50 slurry per sample a minimum of 3 times in 1 mL 1X PBS.
– Resuspend beads in the starting volume and add 30 μL to each tube of lysate (step 1), including no antibody control tubes (step 3), and incubate 1 hour at room temperature.
– Centrifuge on highest speed for 1 minute in a microcentrifuge. Transfer the supernatant to a clean tube using a 26.5-gauge needle and syringe.
– Start the protocol at Step 2.
Materials
MATERIALS
Blotted nitrocellulose membraneLI-CORCatalog #926-31090/926-31092
Odyssey Protein Molecular Weight MarkerLI-CORCatalog #928-40000
Odyssey® Blocking Buffer (PBS) LI-CORCatalog #927-40000 927-40100
Quick Western Kit-IRDye 680RDLI-CORCatalog #926-68100
Protein A Beads Thermo Fisher ScientificCatalog #20333
Anti-ERK2 Mouse AntibodySanta Cruz BiotechnologyCatalog #SC1647
4X Protein Sample Loading BufferLI-CORCatalog #928-40004
20X MES BufferThermo Fisher ScientificCatalog #NP0002
NuPAGE Antioxidant Thermo Fisher ScientificCatalog #NP0005
NuPAGE 10% Bis-Tris gelsThermo Fisher ScientificCatalog #NP0303BOX
IRDye 680RD Goat anti-MouseLI-CORCatalog #926-68070
Safety warnings
See SDS (Safety Data Sheet) for hazards and safety warnings.
Sample Preparation (Day 1)
Sample Preparation (Day 1)
Starting with a total of 820 μg of A431 cell lysate, transfer 200 μg into each of 4 separate 1.5 mL microcentrifuge tubes and place the four tubes on ice.
200 µg A431 cell lysate
Add 5 μL ERK2 antibody to two of the tubes and label as “IP”.
5 µL ERK2 antibody
Add 5 μL 1X PBS to the remaining two tubes and label as “negative control”. Mix all tubes well.
5 µL 1X PBS
Move all four tubes to 4 °C overnight with gentle shaking.
4 °C
Transfer the remaining 20 μg (10 μL) aliquot of A431 lysate to a 1.5 mL microcentrifuge tube.
10 µL A431 lysate
Add an equal volume of 2X diluted Protein Sample Loading Buffer and store at -20 °C until use. Label as “positive control”.
-20 °C Storage
Bead Preparation (Day 2)
Bead Preparation (Day 2)
Add 150 μL of Protein A bead (50:50) slurry to a 1.5 mL microcentrifuge tube and centrifuge 1 minute at highest speed in a microcentrifuge.
00:01:00 centrifugation
150 µL Protein A bead (50:50) slurry
Remove supernatant with a 26.5-gauge needle and 3 mL syringe. Discard supernatant.
Add 1 mL of 1X PBS, making sure the pellet is disrupted, then centrifuge at high speed as in step 7. (1/3)
00:01:00 Centrifugation
1 mL 1X PBS
Add 1 mL of 1X PBS, making sure the pellet is disrupted, then centrifuge at high speed as in step 7. (2/3)
00:01:00 Centrifugation
1 mL 1X PBS
Add 1 mL of 1X PBS, making sure the pellet is disrupted, then centrifuge at high speed as in step 7. (3/3)
00:01:00 Centrifugation
1 mL 1X PBS
Resuspend final pellet in 150μL 1X PBS.
150 µL 1X PBS
Pull Down of Bead:Protein Complex
Pull Down of Bead:Protein Complex
Add 30 μL of bead slurry (prepared in step 11) to each tube (from step 4) and incubate 2 hours at room temperature with rocking on a Nutator™ mixer.
02:00:00 Incubation
30 µL bead slurry
Centrifuge on highest speed for 1 minute to pellet the beads.
Immediately remove the supernatant with a 26.5-gauge needle and 3 mL syringe.
00:01:00 Centrifugation
Wash the beads with 1 mL of 1X PBS each, pellet the beads and remove the supernatant with the needle and syringe. Discard supernatant. (wash 1/4)
1 mL 1X PBS
Wash the beads with 1 mL of 1X PBS each, pellet the beads and remove the supernatant with the needle and syringe. Discard supernatant. (wash 2/4)
1 mL 1X PBS
Wash the beads with 1 mL of 1X PBS each, pellet the beads and remove the supernatant with the needle and syringe. Discard supernatant. (wash 3/4)
1 mL 1X PBS
Wash the beads 1 time with 1 mL of 1X PBS + 0.1% Tween® 20, pellet, and carefully remove ALL liquid from the tube. Discard supernatant. (wash 4/4)
1 mL 1X PBS
Resuspend the beads in 10 µL of 2X diluted Protein Sample Loading Buffer and heat 5 minutes at 100 °C.
00:05:00 heat
10 µL 2X diluted Protein Sample Loading Buffer
100 °C
Cool tubes immediately on ice.
Electrophoresis and Transfer
Electrophoresis and Transfer
Assemble 10% Bis-Tris gel in electrophoresis box.
Add 1X MES-SDS running buffer to entire chamber and 500 µL of antioxidant to inner chamber.
500 µL antioxidant
Centrifuge beads on highest speed for 1 minute, then load the 10 µL of supernatant on a gel.
00:01:00
Note
NOTE: Duplicate sets of samples should be separated by pre-stained MW Markers and should include the positive controls prepared on Day 1 (See Figure 2)
10 µL supernatant
Run gel according to manufacturer’s recommendations.
Transfer the proteins to nitrocellulose using the tank transfer method and 1X Tris-Glycine + 20% methanol buffer for 65 minutes at 100V constant voltage.
01:05:00
Western Blot Workflow
Western Blot Workflow
Note
NOTE: With all Western blotting detection methods, optimal performance will be achieved if blots are allowed to dry prior to detection. Make sure all forceps and incubation boxes have been rinsed with methanol to remove any potential contamination.
Place the membrane in a clean incubation box.
Rinse briefly with 1X PBS. Decant.
Block the membrane in Odyssey® Blocking Buffer for 1 hour at room temperature with shaking.
01:00:00
Using clean scissors, cut the membrane in half and move each half to separate clean incubation boxes.
Note
NOTE: Perform both detection methods simultaneously.
Standard Western Blot Detection
Standard Western Blot Detection
Incubate one blot following the LI-COR standard Western blot protocol using the manufacturer’s recommended amount of ERK2 antibody (1:1000) diluted in Odyssey Blocking Buffer plus 0.2% Tween® 20 for detection.
*For example, add 15 µL of ERK2 antibody to 15 mL Odyssey Blocking Buffer plus Tween 20.
Incubate one hour at room temperature.
01:00:00
Decant antibody, rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (1/4)
00:05:00
Decant the wash, rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (2/4)
00:05:00
Decant the wash, rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (3/4)
00:05:00
Decant the wash, rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (4/4)
00:05:00
Add the IRDye 680RD goat anti-mouse secondary antibody at a 1:15,000 dilution to Odyssey Blocking Buffer + 0.2% Tween 20 and incubate 1 hour at room temperature with shaking.
*For example, add 1 µL of secondary antibody to 15 mL of Odyssey Blocking Buffer plus 0.2% Tween 20.
01:00:00
After 1 hour incubation of the secondary antibody, decant antibody, rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (1/4)
00:05:00 Incubation
Decant the wash, rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (2/4)
00:05:00 Incubation
Decant the wash, rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (3/4)
00:05:00 Incubation
Decant the wash, rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (4/4)
00:05:00 Incubation
Quick Western Blot Detection
Quick Western Blot Detection
Incubate the second half of the membrane following the Quick Western Blot protocol using ERK2 diluted 1:1000 mixed with 1 µL per mL of the Detection Reagent in Odyssey® Blocking Buffer plus 0.2% Tween® 20.
*For example, add 15 µL of ERK2 antibody plus 15 µL of Detection Reagent to 15 mL Odyssey Blocking Buffer plus 0.2% Tween 20. Blot should be incubated at room temperature on a shaker using your standard primary antibody incubation time; in this case, one hour.
01:00:00
Note
NOTE: Please refer to Quick Western Kit-IRDye 680RD pack insert for more detailed instructions on reconstitution and storage of Detection Reagent.
Decant the antibody/Detection Reagent and rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (1/4)
00:05:00
Decant the wash and rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (2/4)
00:05:00 Incubation
Decant the wash and rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (3/4)
00:05:00 Incubation
Decant the wash and rinse briefly one time with 1X PBS + 0.1% Tween 20, then add an excess of wash buffer and incubate 5 minutes with shaking. (4/4)
00:05:00 Incubation
Imaging
Imaging
Scan on Odyssey CLx set to auto, Odyssey Classic at an intensity of 5 for both channels, or Odyssey Sa/Aerius at intensity 7 for both channels.