Introduction

Understanding the spatial and temporal expression patterns of a gene is essential in elucidating its functions. Southwestern blotting is useful in investigating DNA–protein interactions to aid the identification of DNA-binding protein factors. Transcription and hence expression regulation of a gene involves initially highly specific interactions of protein factors with distinct short DNA sequence motifs, 6–8 bp in size, usually located within the promoter or other functional cis-acting elements. Identification of protein factors that bind to genes to turn them on or off is therefore important in investigating gene functions. Southwestern blotting is a technique developed for the detection and characterization of protein factors interacting with distinct DNA motifs. The technique is modified from a previous procedure called 'protein blotting' described by Bowen et al.¹. In this original protocol, proteins are first separated based on size by SDS-PAGE. The polyacrylamide gel containing the proteins is then sandwiched between two filters so that the proteins can diffuse out and bind onto the filters to produce replicas of the gel. One of these filters is used to visualize proteins in the lanes by staining, and the other one is for detecting protein factors interacting with a labeled probe¹. The technique usually requires either heat inactivation² or partial purification^{3, 4, 5} of crude protein extract. Since then, modifications of the initial protocol have been suggested: first, electrophoretic transfer of proteins from the SDS-PAGE gel to the filter is used instead of diffusion so that renaturation of proteins can occur during the transfer process, as the electric field rapidly removes SDS from the proteins; second, before the application of the probe, the filter is incubated with skim milk to block nonspecific and low-affinity binding sites; and finally, DNA binding is performed under optimal salt concentrations to facilitate specific and high-affinity DNA–protein interactions^{6, 7, 8}. A flow chart briefly describing the procedures and time needed for Southwestern blotting is shown in Figure 1.

Figure 1: A flow chart summarizing the approximate time needed and the procedures involved in Southwestern blotting.

Asterisk represents pause points between the steps.

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When studying the interactions between transcription factors with DNA motifs, electrophoretic mobility shift assay (EMSA) is one of the most popular methods as it is rapid, simple and sensitive. The principle behind this technique is that the electrophoretic mobility of the higher molecular weight protein–nucleic acid complex is less than that of the free probe. However, EMSA suffers from many limitations; for example, many transcription factors could bind several related and even unrelated DNA sequences. Also, the electrophoretic mobility shift pattern provides little information regarding the identity of various proteins in the complex(es) and further analysis such as supershift assays are needed. Moreover, supershift assays require prior experience to establish the link between electrophoretic mobility shift pattern and DNA-binding proteins, and also it is difficult to select appropriate antibodies for the study⁹.

Southwestern blotting, on the other hand, could provide information regarding the molecular weights of the DNA-binding proteins as the technique involves the separation of proteins under SDS-PAGE. Hence, with this information, the identity of the protein(s) could be deduced by searching transcription factors databases, for example, TransFac¹⁰. It is, similar to EMSA, also a simple technique that does not require special equipment. The major problem with Southwestern blotting is that the separation of proteins under denaturing SDS-PAGE could dissociate polymeric protein factors leading to ineffective binding of DNA. Therefore, Southwestern blotting is not suitable for detecting protein factors that require more than one subunit for efficient DNA binding. In addition, large quantities of protein extract are required in SDS-PAGE to increase the sensitivity of the technique. Based on the pros and cons of EMSA and Southwestern techniques, a strategy to identify and confirm DNA–protein interactions, with which to study the involvement of DNA motifs and protein factors in gene regulation is suggested in Figure 2.

Figure 2: A flow chart showing a strategy for identifying DNA-binding proteins (e.g., transcription factors) by Southwestern blotting in combination with other methods.

Promoter assays are first performed to identify the promoter regions that are important for regulating transcription of the target gene. Information from promoter studies will be used for designing oligonucleotide primers for electrophoretic mobility shift assay (EMSA), which confirm binding of protein factors with the DNA sequence in an in vitro environment. Southwestern blotting is unique as it is the only technique that shows the molecular weight of the protein factor. On the basis of these studies, one can search databases TransFac and Patch search to identify the protein. To confirm, supershift and chromatin immunoprecipitation (ChIP) assay should then be performed to show the in vitro and in vivo interactions of the protein and the DNA motif using specific antibody.

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Most of the techniques employed in identifying protein factors, when used alone, suffer from some limitations. For example, computer-based analysis, such as P-match search¹¹, often results in suggesting too many possible protein factors. Site-directed mutagenesis coupled to transient expression provides information on functional cis-acting motifs in an in vitro cell model. EMSA shows only if there are factors interacting with a putative motif. Supershift EMSA, on the other hand, serves to confirm some already predicted protein factor(s), as well as it depends on the availability of antibodies and often produces ambiguous data. Hence, it is necessary to combine all these techniques to effectively identify functional protein factors/cis-acting motifs in regulating a gene.

Materials

Reagents

• Tris–hydrochloride (Invitrogen, cat. no. 15506-017)
• HEPES (Sigma, cat. no. H4034)
• Glycerol (USB, cat. no. US16374)
• Magnesium chloride anhydrous (MgCl₂; Sigma, cat. no. M8266)
• Triton X-100 (BDH, cat. no. 306324N)
• DTT (USB, cat. no. 15397)
• PMSF (USB, cat. no. 20203)
• Leupeptin (USB, cat. no. 18413)
• Pepstatin A (USB, cat. no. 20037)
• Aprotinin (USB, cat. no. 11388)
• BSA (Roche Applied Science, cat. no. 10735086001)
• Bio-Rad protein assay (based on Bradford¹²; Bio-Rad, cat. no. 500-0006)
• 30% Acrylamide/bis solution (29:1; Bio-Rad, cat. no. 161-0156)
  Caution Acrylamide and bisacrylamide are neurotoxic and are readily absorbed through skin. Wear gloves to avoid direct contact with the solution.
• Trizma base (Sigma, cat. no. T1503)
• SDS (BDH, cat. no. 108073J)
• Ammonium persulfate (APS; Sigma, cat. no. 248614)
• Tetramethylethylenediamine (TEMED; Invitrogen, cat. no. 15524-010)
• Gly (USB, cat. no. 16407)
• Bromophenol blue (USB, cat. no. 17-1329-01)
• Methanol (Merck KGaA, cat. no. 106009)
• Ready-To-Go T4 polynucleotide kinase (GE Healthcare Life Sciences, cat. no. 27-0737-01)
• Redivue [-³²P] ATP 250 Ci (GE Healthcare Life Sciences, cat. no. AA0068)
  Caution When dealing with radioactive materials, appropriate safety precautions must be followed and National regulations adhered to.
• Skim milk powder (any commercially available low fat milk power is also suitable)
• Oligonucleotides (250 pmol l^{- 1})
• Protein ladder: PageRuler prestained protein ladder (Fermentas, cat. no. SM0671) (other commercially available prestained protein ladders are also suitable)
• Tris-buffered saline (TBS) (see REAGENT SETUP)
• Cell lysis buffer (see REAGENT SETUP)
• Nuclear extract buffer (see REAGENT SETUP)
• Separating gel (see REAGENT SETUP)
• Stacking gel (see REAGENT SETUP)
• Running buffer (see REAGENT SETUP)
• Sample buffer (see REAGENT SETUP)
• Transfer buffer (see REAGENT SETUP)
• TNED buffer (see REAGENT SETUP)
• 10 Oligo annealing buffer (see REAGENT SETUP)

Equipment

• Cell lifter (Corning)
• Centrifuges: Eppendorf 5417R (Eppendorf) and Beckman CS-6R (Beckman)
• Rocker platform (Gyro rocker STR-9; Stuart Scientific) (other 3D gyroscopic rocker or rotation rocker could also be used as an alterative)
• Spectrophotometer (U-2800, Hitachi) (any spectrophotometers which are able to measure absorbance 595 nm are also suitable)
• Vertical electrophoresis apparatus: the Mini-Protean II gel system (Bio-Rad), gel size (8.3 7.3 cm²) (most of the commercially available vertical protein gel electrophoresis systems are also suitable; however, according to the gel sizes of other electrophoresis systems, the volume of buffer or the amount of probe to be used in the procedure should be adjusted)
• Protein transfer system: Mini Trans-Blot Cell (Bio-Rad) (other transfer systems could also be used, but the transfer protocol in Steps 19–21 should be modified accordingly)
• Power supply for SDS-PAGE and electroblotting (POWER PAC300; Bio-Rad) (other power supplies with minimum support of 120 V, 500 mA are also suitable)
• Boiler (any commercially available water boilers are suitable)
• Nitrocellulose membranes (e.g., Hybond-C extra; GE Healthcare Life Sciences) or PVDF membrane (GE Healthcare Life Sciences)
• 3-mm Paper (Whatman)
• MicroSpin G-25 column (GE Healthcare Life Sciences)
• Exposure cassette with intensifying screen (GE Healthcare Life Sciences)
• Autoradiographic film: BioMax MR film (Kodak)

Reagent setup

• TBS 10 mM Tris–Cl, 150 mM NaCl (pH 8.0).
• Cell lysis buffer 20 mM HEPES (pH 7.6), 20% glycerol (vol/vol), 10 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1% triton X-100 (vol/vol), 1 mM DTT, 1 mM PMSF, 10 g ml^{- 1} leupeptin, 10 g ml^{- 1} pepstatin, 100 g ml^{- 1} aprotinin.
  Critical PMSF, leupeptin, pepstatin and aprotinin must be freshly added to the lysis buffer just before use.
• Nuclear extract buffer Cell lysis buffer with 500 mM NaCl.
  Critical PMSF, leupeptin, pepstatin and aprotinin must be freshly added to the nuclear extract buffer just before use.

  	8%	10%	12%
  Water (ml)	2.53	2.03	1.53
  30% Acrylamide (29:1) (ml)	2.00	2.50	3.00
  1 M Tris, pH 8.8 (ml)	2.81	2.81	2.81
  10% SDS (l)	75	75	75
  10% APS (l)	75	75	75
  TEMED (l)	7.5	7.5	7.5

  The volume is enough for single gel (8.3 cm 2 cm; W H)

  Water (l)	2.55
  30% Acrylamide (29:1) (l)	0.625
  1 M Tris, pH 6.8 (l)	0.475
  10% SDS (l)	37.5
  10% APS (l)	37.5
  TEMED (l)	2.5

• Running buffer 20 mM Tris-base, 200 mM Gly, 0.12% SDS (wt/vol)
• Sample buffer 30 mM Tris–Cl (pH 6.8), 30% glycerol (vol/vol), 10% SDS (wt/vol), 1 mM DTT, 0.002% (wt/vol) bromophenol blue
• Transfer buffer 25 mM Tris-base, 190 mM Gly, 20% methanol (vol/vol)
• TNED buffer 10 mM Tris (pH 7.5), 50 mM NaCl, 0.1 mM EDTA and 1 mM DTT
• 10 Oligo annealing buffer 10 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA

Procedure

1. Points from here (point 1) up to and including point 11 are related to Nuclear extract preparationTiming: 2 hCulture cells on a 150-mm diameter dish (see EXPERIMENTAL DESIGN for details). Harvest cells when 80–90% confluency is reached.
   Critical step Avoid overgrowing cells which could lead to apoptosis and affect expression of transcription factors.
2. Remove culture medium and wash cells three times each using 10 ml TBS. In each wash, after adding TBS, shake the dish gently for 1 min before removing the buffer.
3. Add an appropriate amount of cell lysis buffer to the culture dish and proceed to Step 4 immediately. For one 150-mm diameter culture dish, 5-ml cell lysis buffer is recommended.
4. Dislodge the cells by scraping using a cell lifter and transfer the cells into a centrifuge tube.
5. Collect cell nuclei by centrifugation at 2,500g for 5 min at 4 °C. Remove supernatant as much as possible and avoid disrupting of the pellet.
6. Resuspend the pellet in nuclear extract buffer. We recommend using 650 l nuclear extract buffer for a 150-mm diameter dish. Resuspend the pellet gently and completely by pipetting it up and down. Transfer the solution into a 1.5-ml centrifuge tube.
7. Lyse nuclei by rocking the mixture for 1 h at 4 °C on a rocker platform (50 r.p.m.).
8. Remove debris by centrifugation at 12,000g for 10 min at 4 °C.
9. Transfer the supernatant (nuclear extract) into a fresh centrifuge tube.
10. Measure protein concentration of the nuclear extract by a standard protein assay (e.g., Bio-Rad protein assay; see Box 1 and EXPERIMENTAL DESIGN for further details). Use BSA as a standard in the assay.
    Critical step Usually, 2–5 mg ml^{- 1} nuclear extract concentration could be obtained from a 150-mm culture dish with 80–90% confluency depending upon the type of cell. We recommend the concentration should be at least 2 mg ml^{- 1}.
11. Aliquot the extract, quick-freeze the tubes in liquid nitrogen and store at - 80 °C.Pause Point Prepare aliquots of the extract to prevent multiple freezing and thawing. The volume of each aliquot depends upon your experiment. We recommend an aliquot size of 100 l. The extract can be stored at - 80 °C for at least 1 month.
12. Points from here (point 12) up to and including point 18 are related to SDS-PAGETiming: 3 hClean up the gel plates, spacers and comb with 70% ethanol and then rinse thoroughly using distilled water. Set up the apparatus and make sure that there is no leakage according to the manufacturer's manual of the electrophoresis system. If commercially available precast gels are used, proceed to Step 15. Refer to instructions provided by http://www.bio-rad.com for general gel cassette preparation or more specifically while using the Bio-Rad system.
    Caution Glass plates and combs must be thoroughly cleaned.
13. Pour the separating gel mix into the apparatus and overlay the poured gel with isopropanol (500 l). Allow the separating gel to polymerize for 30 min at room temperature (25 °C). In the meantime, prepare the stacking gel mixture as indicated in REAGENT SETUP.
    Critical step TEMED and APS should be added just before pouring the separating gel. The percentage of acrylamide in the separating gel can be adjusted according to the molecular weight of transcription factor.
14. Remove the isopropanol by aspiration, and pour the stacking gel mix into the apparatus. Insert the comb and allow the gel to polymerize for 20 min.Pause Point Polymerized gels can be stored for several hours at room temperature or overnight at 4 °C.
15. Remove the comb, fill the tank with 1 running buffer. Prerun the gel at 100–120 V for 30 min.
16. Mix the loading buffer with 100 g nuclear extract in a 1:1 buffer:sample ratio (vol/vol).
    Critical step The amount of nuclear extract can be adjusted according to the expression level and the affinity of transcription factor with probe.
17. Rinse the wells with 1 running buffer before sample loading as residual unpolymerized acrylamide may affect mobility of proteins in the gel. Boil the samples for 5 min and load the samples and protein ladder into the wells.
18. Run the gel at 100–120 V, until the front dye (bromophenol blue) of the loading buffer reaches the end (2 h). The progress of electrophoresis could be monitored also through the prestained protein ladder.
19. Points from here (point 19) up to and including point 21 are related to ElectroblottingTiming: 1 h 15 minRemove the gel from the gel cassette. Discard the stacking gel. Prepare a nitrocellulose membrane and two 3-mm Whatman papers that are the same size as the separating gel.
20. Set up the transfer as recommended by the manufacturer of the protein transfer system (Bio-Rad).
    Critical step Set up the gel-membrane sandwich under transfer buffer. Bubbles trapped between sandwiched layers must be removed. In addition to Hybond-C membrane, PVDF membrane could also be used as an alternative. However, it is necessary to activate the PVDF membrane by dipping it into methanol before setting up the transfer system. Other electroblotting techniques, such as semi-dry transfer system (Bio-Rad), are also suitable.
21. Electroblot proteins from the gel to the membrane at 100 V at 4 °C for 60 min.
    Critical step After electroblotting, monitor the intensity of the prestained protein ladder on the membrane to estimate the overall transfer efficiency. Proteins with larger molecular weight (>100 kDa) take a longer time to complete the transfer. Increase the transfer time and/or use freshly prepared transfer buffer to enhance the overall efficiency whenever necessary.Pause Point The blot can be stored at 4 °C until the renaturing step.
22. Points from here (point 22) up to and including point 28 are related to Preparation of DNA probeTiming: 1.5 hSynthesize complimentary oligonucleotides containing the functional DNA motif(s) (at least 5-nt long flanking regions on both sides of the motif should be included; totally around 18–30 nt; see EXPERIMENTAL DESIGN section for further details of probe design). Prepare stocks (250 pmol l^{- 1}) of the oligos in distilled water.
23. Mix sense and antisense oligos (20 l each) together. Add 10 oligo annealing buffer (5 l) and distilled water (5 l) to top up the reaction volume to 50 l.
24. Heat to 94 °C for 5 min and gradually cool down the reaction mix to room temperature.
    Critical step Gradual cooling is beneficial to the annealing process. This could be done by denaturing the mix in a boiler. After denaturation, one can turn off the boiler and let the temperature goes down slowly, which should take at least 60 min.Pause Point The annealed oligonucleotides could be stored at 4 °C or frozen until use.
25. Dilute the annealed oligonucleotides into 25 pmol l^{- 1} and use 1 l (i.e., 25 pmol) for labeling reaction.
26. Label the DNA with [-³²P] ATP (50 Ci) by the Ready-To-Go T4 polynucleotide kinase kit according to the manufacturer's instructions (GE Healthcare Life Sciences).
    Critical step Other radioactive labeling methods, such as 5'-end, 3'-end or internal labeling are also suitable.
27. Purify the radioactively labeled DNA probe by a MicroSpin G-25 column to remove the unincorporated radioactive nucleotides.
28. Store the labeled DNA at 4 °C until use.Pause Point The probe should be used as soon as possible or at least within a half-life of ³²P (14 d) when stored at 4 °C. For maximum sensitivity, the labeled DNA should be used within 2 d.
29. Points from here (point 29) up to and including point 32 are related to Renature the protein extract and probe hybridizationTiming: 48 hPlace the blot in a small clear plastic box or hybridization chamber.
30. Renature the proteins in 5 ml TNED buffer containing 5% skim milk overnight at room temperature with shaking (50 r.p.m.). Protect the blot and buffer from exposure of light.
31. Replace the buffer with 3 ml TNED buffer containing 5% skim milk. Add 20 pmol of ³²P-labeled probe (2.0 10⁷ c.p.m.) into the container and shake (50 r.p.m.) the blot overnight at room temperature under light-sealed condition.
32. Wash the membrane three times (15 min each) with TNED buffer at room temperature. After the final wash, seal the membrane by sandwiching it in plastic food wrap.
33. Points from here (point 33) up to and including point 35 are related to AutoradiographyTiming: 1–3 dTo obtain an autoradiogram, insert an X-ray film between the blot (with the gel side face toward the film) in dark room and an intensifying screen and keep the cassette at - 80 °C.
    Critical step The lanes in the blot, in relation to the protein size ladder, could be positioned using phosphorescent ruler, X-ray film marker or tape.
34. Exposure time varies depending upon the specific activity of the probe. Normally exposure time ranges from 1 to 3 d.
    Critical step Intensifying screen is used to reduce the exposure time. Note that all intensifying screens work optimally at - 60 to - 80 °C.
35. Develop, fix and dry the film, according to the manufacturer's or specific laboratory specifications.Troubleshooting

Timing

Steps 1–11, nuclear extract preparation: 2 h
Steps 12–18, SDS-PAGE: 3 h
Steps 19–21, electroblotting: 1 h 15 min
Steps 22–28, preparation of DNA probe: 1.5 h
Steps 29–32, renature the protein extract and probe hybridization: 48 h
Steps 33–35, autoradiography: 1–3 d

Troubleshooting

Troubleshooting advice can be found in Table 1.

Table 1: Troubleshooting table

Full table

Anticipated results

Southwestern blotting is a technique that could provide information on the molecular weight of the protein interacting with specific DNA sequences. With the help of this information, one can dramatically reduce the number of putative transcription factors to be analyzed in a study. Figure 3 shows typical results from Southwestern analysis. Before the experiment shown in Figure 3, synthetic oligonucleotides containing the N1 target motif and a GC-box were designed based on results as suggested in the flow chart (Fig. 2). Southwestern blotting showed two bands on the blot hybridized with the wild-type probe (Fig. 3a), indicating that at least two proteins of sizes 110 and 60 kDa interacted with the probe. To identify the protein specific for the N1 target site, another probe with the N1 motif mutated can be used. After hybridization, only one band with a molecular weight 110 kDa was detected (Fig. 3b), which is consistent with the sizes of GC-box binding Sp proteins (Sp1 and Sp3); thus, it can be established that the N1 target motif interacts specifically with a 60 kDa protein. On the basis of this, the database TransFac can be searched accordingly using the molwSearch utility¹⁰. The identity of the transcription factor binding onto the N1 can be then confirmed by in vitro and in vivo supershift and ChIP assays, respectively, as well as other functional assays⁹.

Figure 3: Southwestern blotting of the N1 motif with SH-SY5Y nuclear extracts.

One hundred microgram of nuclear extract from SH-SY5Y cells was separated in a 10% SDS-PAGE. The separated proteins were transferred to Hybond-C extra membrane, which was then incubated with either the (a) wild-type or the (b) mutated probe, and the signals were visualized by autoradiography. The sequences of the probes are shown below. The underlined sequences indicate the locations of the GC-boxes and the N1 target site. The bold-type sequences are the mutated motifs.

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