A guide to simple, direct, and quantitative in vitro binding assays
- AminoLink Plus coupling resin (Thermo Fischer Scientific, cat. # 20501)
- BupH phosphate buffered saline packs (Thermo Fischer Scientific, cat. # 28372)
- NaOH, 1M (Sigma-Aldrich, cat. #72082)
- Bradford dye (BioRad, cat. # 500-0006)
- Bovine Serum Albumin (BSA) (Sigma-Aldrich, cat. # 9048-46-8)
- HEPES (Sigma-Aldrich, cat. # 7365-45-9)
- Triton X-100 (Sigma-Aldrich, cat. # 9002-93-1)
- Glycerol (Sigma-Aldrich, cat. # G5516)
- Tris Base (Sigma-Aldrich, cat. # 77-86-1)
- NaCl (Sigma-Aldrich, cat. # S3014)
- Sodium dodecyl sulfate (SDS), 20% (w/v), (BioRad, cat. # 1610418)
- Coomassie brilliant blue G-250 (BioRad, cat. # 1610406)
- Methanol, anhydrous (Sigma-Aldrich, cat. # 322415)
- Ammonium Sulfate (Sigma-Aldrich, cat. # 7783-20-2)
- Phosphoric acid (Sigma-Aldrich, cat. # 7664-38-2)
- Bottle top filter, (Sigma-Aldrich, cat. # 430516)
- 2-Mercaptoethanol, (Sigma-Aldrich, cat. # 60-24-2)
- Bromophonol blue (BioRad, cat. # 1610404)
- Amicon Ultra 15 centrifugal filters (Millipore, cat. # UFC903008; cutoff depends on size of protein)
- Coupling buffer: 3.65 × phosphate buffered saline (PBS), pH 7.2, or at NaCl concentration in which the bait protein is stable (i.e. 3.65 × corresponds to 500 mM NaCl).
- Sodium cyanoborohydride, 5M: Dissolve 0.314 g of Sodium cyanoborohydride (NaCNBH3) into a final volume of 1 ml NaOH (1M).
- Quenching buffer (1 M Tris, pH 7.25): Dissolve 60.57 g of Tris base in 300 ml double distilled water (DDW). Adjust pH to 7.25. Adjust volume to 500 ml with DDW.
- Blocking buffer: Dissolve 1g of BSA in 10 ml of quenching buffer (1 M Tris, pH 7.25). Filter-sterilize and store at 4°C.
- Wash solution (1 M NaCl): Dissolve 29.22 g NaCl in 500 ml DDW for a final concentration of 1 M.
- Binding buffer: Dissolve 2.98 g of HEPES in 300 ml DDW. Adjust pH to 7.25. Add 2.92 g NaCl, 50 μl Triton X-100, and 25 ml Glycerol and adjust the volume to 500 ml with DDW. Final concentration is 25 mM HEPES, 100 mM NaCl, 0.01% Triton X-100, and 5% Glycerol, 1 mM DTT.
- Coomassie blue-silver stain : Add 100 ml phosphoric acid to 300 ml DDW. Add 100 g ammonium sulfate, stir until dissolved. Slowly add 1.2 g Coomassie blue G-250. Stir until dissolved (this may take overnight). Add 200 ml methanol and DDW to a final volume of 1 L. Filter through a bottle-top filter.
- Laemmelli sample buffer (LSB), 4 × : Add 4 ml of 20% SDS to 2 ml of 1M Tris, pH 6.8. Add 2 ml glycerol and 0.8 ml of β -mercaptoethanol. Add a small pinch of bromophenol blue. Adjust volume to 10 ml. Stir or rotate until ingredients are well combined. Store aliquots at –20°C.
- End-over-end tube rotator (Argos Technologies, R2001)
- Eppendorf table-top centrifuge at 4°C (Eppendorf, 5417R)
- pH meter (Sartorius, PB-11)
- Spectrophotometer or NanoDrop (Thermo Scientific, NanoDrop 2000c)
- Dry bath incubator (Major Science, MD-01N-110/220)
- Protein electrophoresis system (Bio-Rad, cat. #1658004)
- Gel documentation system (Syngene, InGenius 3)
- ImageJ (National Institute of Health, USA: http://imagej.nih.gov/ij) or a similar quantification software.
- GraphPad Prism (La Jolla, California, USA: http://www.graphpad.com) or a similar data analysis software.
1.Preparation of bait-conjugated beads
1.1.Place 0.5 ml of AminoLink bead slurry (50% beads-50% buffer) in an Eppendorf tube. Keep careful track of the amount of slurry used to be able to reconstitute this ratio later.
1.2.Wash three times with coupling buffer.
1.3.Buffer exchange purified recombinant bait protein into coupling buffer.
1.4.Spin protein at 20000 g, 4°C in a tabletop centrifuge to test protein solubility. Determine protein concentration with a colorimetric assay (i.e. Bradford dye) or by NanoDrop.
1.5.Add purified recombinant bait protein to the washed AminoLink beads. The protein concentration bound to 10 μl of bead slurry should be below the anticipated Kd in a final reaction volume of 500 μl. Save a small amount for subsequent determination of coupling efficiency. Example: to determine the binding affinity of cortactin to Pyk2, we conducted a literature search to obtain values for cortactin binding to other proteins such as Arg kinase . From this search we anticipated a Kd around 0.5 μM. Cortactin has a molecular weight of 61.5 kDa. To obtain a cortactin concentration of 0.1 μM in 500 μl reaction volume, we coupled 3.1 μg of cortactin to 10 μl bead slurry. We used 155 μg cortactin for 0.5 ml of beads.
1.6.Add 5M sodium cyanoborohydride (20 μl/ml) to the reaction slurry (in a chemical hood).
1.7.Rotate end-over-end overnight at 4°C.
1.8.Spin at 8000 g, 4°C for 2 min in a tabletop centrifuge, remove supernatant. Determine binding efficiency by comparing the absorbance of supernatant to input. Binding efficiency should be close to 100%.
1.9.Wash beads three times with quenching buffer.
1.10.To block the remaining active sites on the beads, add 1 ml quenching buffer containing 100 mg/ml BSA, and 5M sodium cyanoborohydride (20 μl/ml).
1.11.Block beads overnight (or for a minimum of 6 to 8 h).
1.12.Centrifuge at 8000 g, 4°C and remove buffer by aspiration. Take care not to aspirate any beads.
1.13.Wash at least four times with wash solution and monitor for complete removal of uncoupled BSA with Bradford dye. Wash until no protein is detected in the wash solution. Add binding buffer to make a 50%-50% ratio of bead to buffer slurry.
1.14.To make BSA control beads, start at the blocking step (step 1.10) using AminoLink beads that were washed with blocking buffer.
2.Preparation of prey protein solution
2.1.Purify the desired protein according to your standard protocol.
2.2.Buffer exchange the protein into binding buffer.
2.3.Concentrate the prey protein using an Amicon centrifugal filter with appropriate molecular weight cut-off.
2.4.Determine if the protein is stable under these conditions.
2.4.1.Spin the protein at 20000 g, 4°C for 10 min. Visually inspect the tube for the presence of a large protein pellet.
2.4.2.Pre-clear the protein by adding 20 μl BSA beads per each ml of protein sample. Rotate end- over-end for 30 min at 4°C.
2.5.Spin down the beads for five minutes at 14000 g, 4°C. Carefully remove and keep the supernatant containing the protein in solution.
2.6.Determine protein concentrations before and after steps 2.4.1 and 2.5. If a significant amount of protein is lost, adjust buffer conditions to stabilize the protein .
3.Screening for binding conditions
3.1.Prepare four dilutions of the protein in solution (in a total volume of 500 μl each). Highest concentration should be approximately five times the expected Kd. The lowest concentrations should be about 0.5 × the expected Kd. Add 10 μl of bead slurry containing the bait protein.
3.2.Repeat this process with 10 μl of BSA control beads instead of protein beads.
3.3.Rotate end-over-end at 4°C for one hour.
3.4.Spin down beads for two minutes at 8000 g, 4°C.
3.5.Carefully remove supernatant and transfer to a fresh tube.
3.6.Wash twice quickly with binding buffer.
3.7.After the last wash, aspirate the entire wash buffer without disturbing the beads. Use a 27G × 1/2 needle attached to 1 ml syringe to aspirate the remaining traces of buffer.
3.8.Add 40 μl of LSB and boil samples for 10 min. Alternatively, samples can be incubated on a heat block at 95°C for 10 min. This step will disrupt the binding between bait and prey protein. The bait protein will remain covalently bound to the beads, whereas the prey protein will be released to the supernatant.
3.9.Spin samples for 5 min at 20000 g in a tabletop centrifuge to precipitate bait-conjugated beads.
3.10.Load 35 μl of sample per well on an SDS-PAGE gel. Load supernatant only. Add the same volume in each lane and avoid loading any beads. Run the gel and stain with Coomassie blue-silver overnight.
3.11.De-stain gels completely by washing several times with DDW until no dye comes out of the gel. This may take several hours. Prey protein should be visible in samples pulled-down by bait protein beads, but not by BSA control beads (Fig. 3B and 3C).
4.Performing the binding assay
4.1.Make serial dilutions of the prey protein. The highest concentration should be about 5–10 times the expected Kd, with two to three points below the expected Kd. Concentrations will range from 0 μM to five to ten times the expected Kd, nine samples total, in a volume of 490 μl.
4.2.Add 10 μl of AminoLink beads attached to bait protein, or BSA control beads. Rotate end-over-end for one hour at 4°C.
4.3.Proceed as in Section 3.3 to 3.9 above. Load samples that were pulled-down with bait protein beads on a different gel from samples pulled-down with BSA beads.
4.4.Stain gels overnight using sufficient amount of Coomassie blue-silver staining solution.
4.5.De-stain gels completely using DDW until no color comes out of the gels.
5. Gel analysis
5.1.Analyzing the gels using ImageJ:
5.1.1.Scan the gels after complete destaining using a gel scanner.
5.1.2.Open images in ImageJ and invert the images (shift + control + I).
5.1.3.Draw a box around the region of interest (i.e. the highest concentration band).
5.1.4.Select “analyze - measure” (control + M). A result window containing lane number, area, mean, min, and max values will open. The relevant value is the mean density of the band.
5.1.5.Move the box to the next band and measure band density in the same way. Repeat this until all band densities are measured (Fig. 4B).
5.2.Analysis of binding data and determining the dissociation constant using GraphPad Prism:
5.2.1.For a single binding experiment, select “file—new—new project file”. Select “XY” under “new table and graph”. Under “enter/import data” select: “Y: enter and plot single Y value for each point”. In the spreadsheet, fill in the following data:
5.2.2.On the top toolbar under “analyze” click on the option “fit curve with nonlinear regression”. A window will appear asking you how to analyze the data. Under the “fit” tab select “binding saturation—one site specific binding”.
5.2.3.At the left side of the screen, under “results”, a window of “one site—specific binding” data will appear, as in Figure 4E. The first set of values is your actual results (best-fit values). The second set is your standard error (std. error), followed by the 95% confidence intervals. For example, from the table in Figure 4E, you can deduce that your Kd is 0.6317 ± 0.08976 μM. You can say with 95% confidence that the Kd falls between 0.4194 to 0.8440. The R2 value is 0.9839, which indicates a very good curve fit. Look at the graph obtained from your data and convince yourself that you have a good graph with saturated data and sufficient points around the Kd and at the slope.
5.3.Repeating experiments and combining data: Experiments need to be repeated at least three times in order to combine data and obtain a final Kd value.
5.3.1.In GraphPad, select “file–new–new project file”.
5.3.2.Under “tables and graphs” select “XY”.
5.3.3.Select “enter/import data” and enter 3 replicate values in side-by-side sub-columns. Perform the analysis as described in 5.2 above for the single experiments. You will now get a graph with combined data and error bars (Fig. 4F).
|1.8||Low coupling efficiency of bait protein to beads||Sample buffer contains primary amines||
|2.2, 2.4||Prey protein not soluble at high concentrations in binding buffer||Buffer conditions not compatible with protein||
|2.3||Protein precipitates during concentration with Amicon centrifugal filters||
|3.11||Prey protein binds to BSA beads||Protein is not stable||
|3.11||Prey protein is degraded||Protein is not stable||
|3.11||Variation in Kd between experimental repeats||Binding efficiency varies due to temperature and buffer conditions||
|3.11, 4.5, 5.2||Binding does not increase with increased protein concentration||Binding may be saturated||
|3.11, 4.5, 5.2||Binding pattern erratic||Bead volume not even between different reaction tubes||
|3.11, 4.3||SDS-PAGE gels expand, uneven running of gels||Beads loaded on gel||
|3.11||No binding to bait protein beads||Concentration of bait or prey protein is too low||
|3.11||Bait protein does not bind||No interactions between bait and prey protein||
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