Far Western Blotting

Far western blotting is a method for characterizing protein-protein interaction. Protein samples are first separated by gel electrophoresis, it is immobilized on a membrane. There it is probed with a non-antibody protein. The probe is directly attached to denatured/seperated protein, immobilized on a membrane. Mostly far western blotting detects detect direct interaction. Far-western assay is often used to confirm direct interaction following immunoprecipitation or assays.

The limitation of far-western starts with its own principle of direct interaction. Only a certain n umber of proteins which show direct interaction can only be detected. Proteins present in the cell lysate are denatured in the gel electrophoresis, If native protein is required for direct proteinprotein interaction, it becomes a limitation. Thus far-western blotting is applied only for domains that bind to short,linear peptide motifs. Signaling in protein interaction is done via modular domain-peptide interaction thus far western interaction is used for signaling networks. Either the whole protein or peptide sequence containing a suspected binding interface is used to interacting partners immobilized on a membrane. The interaction is visualised by direct labelling of the probe or by adding addition of specific antibodies.

Considerations for selecting probe

  1. i) A probe should be easy to purify. Expressing probe proteins ae generally used for this purpose. This probe should be short in length as proteins more than 100 kDa is not soluble. So fragment of protein containing only the known protein is considered instead of whole protein.
  2. II) For purification purpose the probe should be easy to fuse. Generally, the probes are fused with gluta-S-thione transferases (GST) fusions. The advantage of GST fusion is it is easier to purify using glutathione columns and detection is done by anti-GST antibodies. GST exits as a stable dimer in solutions, it works as an added advantage.

Application

Far-western blot can detect a protein based on the presence of presence of peptide sequence without any future knowledge of the protein. From the intensity of the bands formed in the far-western blot the number and relative affinity of the binding partners of the probe can be detected. Some proteins bind with probes after post translational modifications. For those proteins, far-western blotting can be used to access the modification state of these proteins.

The following procedure is to demonstrate the usage of far-western blotting. GST- tagged Src homology (SH2) domains which bind specifically to tyrosine-phosphorylated target proteins. It is used to probe the tyrosine phosphorylation of cellular proteins.

Materials

Subcloning of GST-SH2 Construct

  1. pGEX-6P1 vector.
  2. Luria-Bertani (LB)-ampicillin agar plate: LB agar plate with 100 μg/ mL ampicillin.
  3. Pfu DNA polymerase.
  4. Custom oligonucleotide primers.
  5. Competent bacteria (strain NB42 or DH5 α ).

Evaluation of GST-SH2 Clones

  1. LB-ampicillin: LB broth with 50 μ g/mL ampicillin.
  2. Isopropyl- β -d-thiogalactoside (IPTG).
  3. Bacteria Triton X-lysis buffer (BXB): Phosphate buffered saline (PBS) with 100 mM ethylene diamine tetraacetic acid (EDTA), 1% Triton X-100; add phenylmethyl sulphonyl fluoride (PMSF) to 1 mM, aprotinin to 1% v/v (3 trypsin international units (TIU)/mL), dithiothreitol (DTT) to 1 mM just before use.
  4. Sonicator with microtip probe.
  5. 5X sample buffer: 0.3 M Tris–HCl pH 6.8, 10% sodium dodecyl sulfate (SDS), 25% β -mercaptoethanol, 0.1 mg/mL bromophenol blue, 45% glycerol.
  6. Glutathione Sepharose 4B.
  7. 12% SDS-polyacrylamide gel electrophoresis (PAGE) mini gel.
  8. Control lysates.
  9. Antiphosphotyrosine antibody.
  10. Coomassie Blue solution: 40% methanol, 10% acetic acid, 0.25% Coomassie Blue R-250.
  11. Fix solution: 20% methanol, 10% acetic acid.
  12. Bacteria stock solution: 50% glycerol (autoclaved).
  13. Cryogenic tubes.

Large-Scale Preparation of GST-SH2 Probe

  1. Tris–NaCl–EDTA (TNE) buffer: 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 10 mM EDTA; add aprotinin to 1% (3 TIU/ mL), PMSF to 1 mM just before use.
  2. Chromatography column.
  3. Elution buffer: 20 mM reduced glutathione, 100 mM Tris– HCl, pH 8.0; add aprotinin to 1% (3 TIU/mL), PMSF to 1 mM just before use.
  4. Sephadex G-25 PD-10 column.
  5. Dialysis membrane tubing.
  6. PBS with 10% glycerol.
  7. Bio-Rad Bradford dye reagent.
  8. Ultrafiltration membrane.

Far-Western

  1. Kinase lysis buffer (KLB): 150 mM NaCl, 25 mM Tris–HCl pH 7.4, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% sodium pyrophosphate, 10 mM β -glycerophosphate, 10 mM sodium fluoride (NaF); add aprotinin to 1% (3 TIU/mL),

PMSF to 1 mM, pervanadate (50 mM orthovanadate, 4% hydrogen peroxide) to 50 μ M just before use.

  1. Sodium orthovanadate: dissolve powdered sodium orthovanadate (final concentration will be 50 mM, but leave some extra volume for multiple rounds of pH adjustment); adjust with NaOH to pH of 10 (solution will turn bright yellow); boil in microwave until colorless, then stir until cooled to room temperature; adjust pH once again to 10, and repeat boiling; continue boiling and adjusting pH as above until pH stays at 10 after boiling (usually three rounds total); adjust volume for 50 mM, filter, and store at room temperature (RT).
  2. Pervanadate solution: mix 16- μ L concentrated hydrogen peroxide and 100 μ L 50 mM sodium orthovanadate; incubate at RT for 30 min (not stable, needs to be freshly prepared before use).
  3. Positive control lysates: equal amounts of KLB lysate from pervanadate-treated NIH 3T3, HepG2, A431, and MR20 cells were combined.
  4. Negative control lysate: lysates of each cell line were prepared in the absence of vanadate, combined, and treated with tyrosine phosphatase PTP-1B for 1 h at RT.
  5. 12% SDS-PAGE gel (1.0 mm thickness, for three mini-gels). Resolving gel is preapared in the follwing composition: Acrylamide/bis- acrylamide- 20 mL, 1-M Tris-HCl (pH 8.8)-18.6 mL, Distilled water 10.5 mL, 10% SDS 500 μL, 10% Ammonium persulfate 500 μL, TEMED 16.7 μL. Stacking gel is preapared in the following composition: Acrylamide/bis- acrylamide- 2.2 mL, 1-M Tris-HCl (pH 6.8)-2.1 mL, Distilled water 12.2 mL, 10% SDS 167 μL, 10% Ammonium persulfate 125 μL, TEMED 16.7 μL.
  6. Electrophoresis buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS.
  7. Nitrocellulose membrane (0.2- μ m pore).
  8. Transfer buffer: 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), pH 11, 20% methanol, kept at 4°C.
  9. Tris buffered saline-Tween 20 (TBST): 25 mM Tris–HCl, pH 8, 150 mM NaCl, and 0.05% v/v Tween-20.
  10. Blocking solution: 10% fat-free milk in TBST, 1 mM EDTA, 1 mM sodium orthovanadate.
  11. Labeling reagent: Glutathione–HRP conjugate or anti-GST–HRP conjugate .
  12. Chemiluminescence kit
  13. Imaging: X-ray film; CCD detection system.

Stripping and Reprobing

  1. Acidic stripping buffer: 100 mM Glycine–HCl pH 2.0.
  2. SDS-ME stripping buffer: 2% SDS, 100 mM β -Mercaptoethanol, 62.5 mM Tris–HCl pH 6.8.

Methods

Protein molecules are separated by SDS-PAGE and transferred to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. The blocked membrane is then incubated with a probe followed by appropriate wash and bound probes are visualized. All protein samples which are compatible with western blotting can be used. It includes whole cell lysates, purified proteins and native or denatured proteins. In far western blotting probing is single step process. It does not have denaturation and renaturation procedures. The probe is labelled directly. Far western blotting can be optimized to detect invitro between modular binding domain probes and immobilized proteins containing short peptide motifs.

A specific protocol for generating GST-SH2 domain probes and using them to probe tyrosine-phosphorylated whole cell lysates. These procedures can be adapted for any modular protein binding domain and its binding partners with minor modifications.

Detection of a particular signal is strongly dependent on the quality of the probe protein. When a probe is used the following considerations should be followed

  1. i) The purified probe is siolublem folded and not significantly degraded.
  2. ii) Activity of the probe and binding conditions should be optimized.

iii) Positive and negative controls should always be included.

A detailed protocol for generation and evaluation of GST-SH2 fusion probes for the above considerations.

Subcloning of GST-SH2 Construct

  1. Retrieve cDNA and protein sequences of a SH2 domain containing protein of interest, e.g., at NCBI Entrez Gene.
  2. Find location of the SH2 domain using the protein sequence at ScanProsite.
  3. Find the nucleotide sequence corresponding to the SH2 domain using the sequence editor program.
  4. Find academic or industry source for corresponding cDNA, otherwise clone the cDNA by RT-PCR method.
  5. Design primers for PCR.
  6. Amplify the SH2 fragment by PCR using the oligonucleotide primers and the cDNA template.
  7. Digest the PCR product, and then purify the fragment from an agarose gel.
  8. Insert the purified SH2 fragment into a pGEX vector digested with appropriate restriction enzymes.
  9. Transform competent bacteria and grow overnight on LBampicillin agar plate.

Evaluation of GST-SH2 Clones

Protein expression and solubility of GST-SH2 clones can be tested quickly in small-scale bacterial cultures, and the activity of the probe can be tested at the same time by a control pull-down assay.

  1. Inoculate 0.4 mL dense liquid culture of bacteria into 1.6 mL fresh prewarmed LB-ampicillin.
  2. Shake at 37°C for 1 h.
  3. Add IPTG to 0.1 mM and shake at 37°C for 1 h to induce protein expression.
  4. Transfer 1.5 mL bacteria to microcentrifuge tubes and spin at 10,000 × g at 4°C for 2 min in microfuge.
  5. Remove supernatant and resuspend bacterial pellet in 0.4 mL BXB.
  6. Vortex to resuspend, then sonicate briefly (e.g., 2–5 s at relatively low power) on ice to break cells, let sit on ice, then repeat. Try to avoid foaming; if foaming occurs, let rest on ice for a few minutes to allow foam to dissipate.
  7. Remove 10 μ L of total lysate, add 2.5 μ L 5× sample buffer for gel.
  8. Spin rest of lysate for 5 min in microfuge at 10,000 × g at 4°C, transfer supernatant to a new tube.
  9. Take 10 μ L of the cleared lysate for gel as above.
  10. Take 100 μ L of cleared lysate and add to 10 μ L glutathioneagarose bead slurry (cut end off pipet tip with razor blade to more accurately pipet beads).
  11. Rotate at 4°C for 30 min.
  12. Spin out briefly, and wash beads 3× with 1 ml cold BXB.
  13. Resuspend the bead pellet with BXB, take 10 μl, and add 2.5 μl 5× sample buffer for gel.
  14. Boil all samples and run on 12% SDS gel.
  15. When gel is done, stain for 15 min with Coomassie blue solution, destain with fix solution, and then dry.

Steps 16–19 : Evaluate pTyr binding activity by GST pull-down assay (optional)

  1. Incubate remaining beads (GSH-bound fractions for pGEXSH2 clones and a control pGEX clone, if any) with 10 μ g cell lysates.
  2. Rotate for 1 h at 4°C, and wash three times with BXB.
  3. Boil all samples and run on 12% SDS gel.
  4. Perform western blotting with anti-phosphotyrosine antibody.
  5. Subject positive clones to DNA sequencing and store bacteria in 25% (v/v) sterile glycerol at −70°C.

 Large-Scale Preparation of GST-SH2 Probe

GST-SH2 probe is purified following the standard protocol for preparation of GST fusion proteins using pGEX series bacterial expression vectors.

  1. Inoculate frozen stock culture of a verified GST-SH2 clone in 50-mL LB-ampicillin overnight.
  2. Inoculate 50 mL of dense overnight culture to 1 L LB-ampicillin. Shake at 37°C for 2 h.
  3. Add IPTG to 0.1 mM. Shake at 37°C for 3 h.
  4. Centrifuge at 5,000 × g at 4°C for 10 min.
  5. Resuspend pellet with in 5–20 mL ice-cold BXB, transfer to 50-mL tube, and sonicate on ice until cells are broken.
  6. Centrifuge at 5,000 × g at 4°C for 10 min to remove debris.
  7. Add glutathione-agarose to supernatant: 3 mL bead slurry per liter original culture.
  8. Rotate at 4°C about 1 h (up to 2 h).
  9. Wash beads with TNE buffer: spin out beads at low speed, remove supernatant, and resuspend beads in fresh buffer. Repeat 3–5 times.
  10. To elute GST-SH2, pour beads into small disposable column, elute with approximately three bead volumes of elution buffer.
  11. Change buffer by gel filtration on a Sephadex G-25 PD-10 column according to the supplier’s instructions. Briefly, equilibrate column with approximately 25 mL PBS-10% glycerol. Discard the flow-through. Add sample followed by buffer up to a total volume of 2.5 mL. Discard the flowthrough. Elute with 3.5 mL buffer (collect seven 0.5 mL fractions of the eluate in separate tubes.
  12. Estimate relative protein concentration with dye reagent, combine top three fractions into one tube..
  13. Determine protein concentration by Bradford assay, take 500 μ g of protein and dilute to 0.1 μ g/ μ L with PBS-10% glycerol, and aliquot 50 μ L diluted probe into chilled microcentrifuge tubes. Store the aliquots and the undiluted probes at −70°C.
  14. Evaluate the purification fractions by 12% SDS gel.

Far-Western Blotting

  1. Separate proteins on SDS-polyacrylamide gels and transfer to nitrocellulose or PVDF membranes following general western blotting protocol.
  2. Block membranes in blocking solution for about 1 h at room temperature or at 4°C overnight.
  3. To label probe, thaw the stored probe on ice and add 5 μ L GSH–HRP conjugate (0.1 μg/ μl) to 50 μl of diluted probe (0.1 μg/ μl).
  4. Incubate on ice for about 1 h. Labeled probes can be storedat 4°C.
  5. Dilute labeled probe to optimal concentration with blocking buffer and apply to the blocked membrane.
  6. Let probe bind for 1–2 h at RT, then wash with multiple changes TBST for 20 min.
  7. Visualize signal by enhanced chemiluminescence according to manufacturer’s instruction.
  8. Take appropriate exposure using X-ray films or an image analyzer e.g., Kodak Image Station system.

Stripping and Reprobing

Generally, fresh membranes are best for far-western blotting; stripping and reprobing of the membrane may result in significant signal loss and increased nonspecific background. Nevertheless recycling membranes might be beneficial if sample is limited, or if precise comparison of specific bands is needed within the same membrane.

  1. After initial probing, keep membrane wet; it can be stored wet, wrapped in plastic wrap, at 4°C (up to a week) or −20°C (for longer period).
  2. Rinse the membrane twice with TBST.
  3. Immerse membrane in stripping buffer at RT for 20 min under gentle rocking agitation.
  4. Wash membrane in large volume of TBST at RT for 45 min with frequent shaking.
  5. Proceed to blocking and reprobing.