Survival of Probiotic Bacterial Cells in the Upper Gastrointestinal Tract and the Effect of the Surviving Population on the Colonic Microbial Community Activity and Composition.

Many health-promoting effects have been attributed to the intake of probiotic cells. However, it is important that probiotic cells arrive at the site of their activity in a viable state in order to exert their beneficial effects. Careful selection of the appropriate probiotic formulation is therefore required as mainly the type of probiotic species/strain and the administration strategy may affect survival of the probiotic cells during the upper gastrointestinal (GIT) passage. Therefore, the current study implemented Simulator of the Human Microbial Ecosystem (SHIME®) technology to investigate the efficacy of different commercially available probiotic formulations on the survival and culturability of probiotic bacteria during upper GIT passage. Moreover, Colon-on-a-Plate (CoaP™) technology was applied to assess the effect of the surviving probiotic bacteria on the gut microbial community (activity and composition) of three human donors. Significantly greater survival and culturability rates were reported for the delayed-release capsule formulation (>50%) as compared to the powder, liquid, and standard capsule formulations (<1%) (p < 0.05), indicating that the delayed-release capsule was most efficacious in delivering live bacteria cells. Indeed, administration of the delayed-release capsule probiotic digest resulted in enhanced production of SCFAs and shifted gut microbial community composition towards beneficial bacterial species. These results thus indicate that careful selection of the appropriate probiotic formulation and administration strategy is crucial to deliver probiotic cells in a viable state at the site of their activity (distal ileum and colon).

Association of dietary flavan-3-ol intakes with plasma phenyl-γ-valerolactones: analysis from the TUDA cohort of healthy older adults.

BACKGROUND: Dietary polyphenols, including flavan-3-ols (F3O), are associated with better health outcomes. The relationship of plasma phenyl-γ-valerolactones (PVLs), the products of colonic bacterial metabolism of F3O, with dietary intakes is unclear. OBJECTIVES: To investigate whether plasma PVLs are associated with self-reported intakes of total F3O and procyanidins+(epi)catechins. DESIGN: We measured 9 PVLs by uHPLC-MS-MS in plasma from adults (>60y) in the Trinity-Ulster-Department of Agriculture (TUDA study (2008 to 2012; n=5186) and a follow-up subset (2014 to 2018) with corresponding dietary data (n=557). Dietary (poly)phenols collected by FFQ were analyzed using Phenol-Explorer. RESULTS: Mean (95% confidence interval [CI]) intakes were estimated as 2283 (2213, 2352) mg/d for total (poly)phenols, 674 (648, 701) for total F3O, and 152 (146, 158) for procyanidins+(epi)catechins. Two PVL metabolites were detected in plasma from the majority of participants, 5-(hydroxyphenyl)-γ-VL-sulfate (PVL1) and 5-(4'-hydroxyphenyl)-γ-VL-3'-glucuronide (PVL2). The 7 other PVLs were detectable only in 1-32% of samples. Self-reported intakes (mg/d) of F3O (r = 0.113, P = 0.017) and procyanidin+(epi)catechin (r = 0.122, P = 0.010) showed statistically significant correlations with the sum of PVL1 and PVL 2 (PVL1+2). With increasing intake quartiles (Q1-Q4), mean (95% CI) PVL1+2 increased; from 28.3 (20.8, 35.9) nmol/L in Q1 to 45.2 (37.2, 53.2) nmol/L in Q4; P = 0.025, for dietary F3O, and from 27.4 (19.1, 35.8) nmol/L in Q1 to 46.5 (38.2, 54.9) nmol/L in Q4; P = 0.020, for procyanidins+(epi)catechins. CONCLUSIONS: Of 9 PVL metabolites investigated, 2 were detected in most samples and were weakly associated with intakes of total F3O and procyanidins+(epi)catechins. Future controlled feeding studies are required to validate plasma PVLs as biomarkers of these dietary polyphenols.

Vitamin B-6 and riboflavin, their metabolic interaction, and relationship with MTHFR genotype in adults aged 18-102 years.

BACKGROUND: The generation of the active form of vitamin B-6, pyridoxal 5'-phosphate (PLP), in tissues is dependent upon riboflavin as flavin mononucleotide, but whether this interaction is important for maintaining vitamin B-6 status is unclear. OBJECTIVE: To investigate vitamin B-6 and riboflavin status, their metabolic interaction, and relationship with methylenetetrahydrofolate reductase (MTHFR) genotype in adulthood. METHODS: Data from 5612 adults aged 18-102 y were drawn from the Irish National Adult Nutrition Survey (NANS; population-based sample) and the Trinity-Ulster Department of Agriculture (TUDA) and Genovit cohorts (volunteer samples). Plasma PLP and erythrocyte glutathione reductase activation coefficient (EGRac), as a functional indicator of riboflavin, were determined. RESULTS: Older (≥65 y) compared with younger (<65 y) adults had significantly lower PLP concentrations (P < 0.001). A stepwise decrease in plasma PLP was observed across riboflavin categories, from optimal (EGRac ≤1.26), to suboptimal (EGRac: 1.27-1.39), to deficient (EGRac ≥1.40) status, an effect most pronounced in older adults (mean ± SEM: 76.4 ± 0.9 vs 65.0 ± 1.1 vs 55.4 ± 1.2 nmol/L; P < 0.001). In individuals with the variant MTHFR 677TT genotype combined with riboflavin deficiency, compared with non-TT (CC/CT) genotype participants with sufficient riboflavin, we observed PLP concentrations of 52.1 ± 2.9 compared with 76.8 ±0.7 nmol/L (P < 0.001). In participants with available dietary data (i.e., NANS cohort, n = 936), PLP was associated with vitamin B-6 intake (nonstandardized regression coefficient ß: 2.49; 95% CI 1.75, 3.24; P < 0.001), supplement use (ß: 81.72; 95% CI: 66.01, 97.43; P < 0.001), fortified food (ß: 12.49; 95% CI: 2.08, 22.91; P = 0.019), and EGRac (ß: -65.81; 95% CI: -99.08, -32.54; P < 0.001), along with BMI (ß: -1.81; 95% CI: -3.31, -0.30; P = 0.019). CONCLUSIONS: These results are consistent with the known metabolic dependency of PLP on flavin mononucleotide (FMN) and suggest that riboflavin may be the limiting nutrient for maintaining vitamin B-6 status, particularly in individuals with the MTHFR 677TT genotype. Randomized trials are necessary to investigate the PLP response to riboflavin intervention within the dietary range. The TUDA study and the NANS are registered at www.ClinicalTrials.gov as NCT02664584 (27 January 2016) and NCT03374748 (15 December 2017), respectively.Clinical Trial Registry details: Trinity-Ulster-Department of Agriculture (TUDA) study, ClinicalTrials.gov no. NCT02664584 (January 27th 2016); National Adult Nutrition Survey (NANS), ClinicalTrials.gov no. NCT03374748 (December 15th 2017).

Southwestern Blotting Assay.

Southwestern blotting is a technique used to study DNA-protein interactions. This method detects specific DNA-binding proteins by incubating radiolabeled DNA with a gel blot, washing, and visualizing through autoradiography. A blot resulting from 1-dimensional SDS-PAGE reveals the molecular weight of the binding proteins. To increase separation and determine isoelectric point a 2-dimensional gel can be blotted. Additional dimensions of electrophoresis, such as a gel shift (EMSA), can precede isoelectric focusing and SDS-PAGE to further improve separation. Combined with other techniques, such as mass spectrometry, the DNA-binding protein can be identified.

Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach.

Gene network of a phosphoglycerate mutase in muscle wasting in mice.

We previously identified the insertion of an intracisternal A-particle retrotransposons (IAPs) sequence in a gene, 9630033F20Rik, that contains domains involved in glycolysis from a mouse model called lethal wasting (lew). However, because both IAP insertion and the muation of vesicle-associated membrane protein 1 (VAMP1) were discovered from lew, the impact of the IAP insertion and Vamp1 on the lew mouse phenotype needs further investigation. In this study, the effect of the 9630033F20Rik and Vamp1 on glycolysis and muscle-wasting genes in heart, muscle, and brain tissues was further investigated using data of gene expression profiles in these tissues. Our data indicated that the expression levels of 9630033F20Rik and Vamp1 are not associated with each other. While 9630033F20Rik affects the expression of several key genes in pathways of glycolysis and muscle wasting, Vamp1 affects a different set of genes, with fewer numbers. In situ hybridization indicated that the expression of 9630033F20Rik is different in musculoskeletal tissues between the muscle-wasting mouse model and the wild-type model. Our data indicated that 9630033F20Rik may play an important role in muscle wasting and that it has a distinguished characterization of gene network. Our data also suggest that both 9630033F20Rik and Vamp1 play functional roles in muscle development and lead to the muscle-wasting phenotype when they are mutated.

T(3): targeted proteomics of DNA-binding proteins.

A technique that allows the inclusion of a specific DNA to enrich and direct proteomic identification of transcription factors (TFs) while providing a route for high-throughput screening on a single platform would be valuable in investigations of gene expression and regulation. Polyvinylpyrrolidone binds DNA avidly while binding negligible amounts of protein. This observation is used in a proof-of-concept method to enrich for TFs by combining nuclear extract with a specific DNA sequence and immobilizing the DNA-protein complex on a polyvinylpyrrolidone (PVP)-coated MALDI (matrix-assisted laser desorption/ionization) plate. Any unbound proteins are washed away and further processed for analysis in a MALDI-TOF/TOF (tandem time-of-flight) mass spectrometer. Enrichment on a PVP-coated plate gives the unique advantage of purification, enzymatic digestion, and analysis on a single platform. The method is termed T(3) because it combines Targeted purification on a Target plate with Targeted proteomics. Validation was achieved in model experiments with a chimeric fusion protein, green fluorescent protein-CAAT enhancer binding protein (GFP-C/EBP), with an oligonucleotide containing the CAAT sequence. Both domains were identified with an expectation value of less than 10(-15) and more than 15% sequence coverage. The same oligonucleotide mixed with HEK293 cell nuclear extract allowed the unambiguous identification of native human C/EBP alpha with 24.3% sequence coverage.

Promoter trapping method: transcription factor purification using human telomerase reverse transcriptase promoter.

BACKGROUND: Transcription factors bind to response elements on the promoter regions of genes to regulate transcriptional activity. One of the major problems with identifying transcription factors is their low abundance relative to other proteins in the cell. Developing a purification technique specific for transcription factors is crucial to the understanding of gene regulation. Promoter trapping is a method developed that uses the promoter regions as bait to trap proteins of interest and then purified using column chromatography. Here we utilize this technique to study the telomerase promoter, which has increased transcriptional activity in cancer cells. Gaining insight on how to control the enzyme at the promoter level may give new routes towards cancer treatments. RESULTS: Our findings show that the telomerase promoter (-170 - +91) and Promoter Trapping isolate a transcriptionally active and reproducible complex, when analyzed by liquid chromatography tandem mass spectrometry. We were also able to identify transcription factors, including AP-2 and SP1 known to bind this promoter, as well as show that these two proteins can bind to each other's response element. CONCLUSION: Here we focus on verifying the ability and versatility of Promoter Trapping coupled with additional well-characterized methods to identify already known factors responsible for telomerase transcriptional regulation.

Coupling of deoxyribonucleic acid to solid supports using 3' terminal ribose incorporation.

To develop a new form of DNA coupling under mild reaction and coupling conditions, DNA oligonucleotides were synthesized containing a 3' ribonucleotide. Upon reaction with millimolar sodium metaperiodate (NaIO4), the ribose is oxidized to a dialdehyde at pH 6.8. This reaction is complete in 30min, is quenched with millimolar sodium metabisulfite (Na2S2O5) and is then suitable for coupling to hydrazide-agarose supports. Coupling occurs with a half-time of 27min and 80% couples in 2h. The EP18 oligonucleotide which binds to the CAAT enhancer binding protein (C/EBP) was synthesized with a 3' ribose (rEP18) and coupled to hydrazide-agarose. The columns prepared show no significant loss of the oligonucleotide after 50 days. A crude bacterial extract from cells expressing a chimeric fusion protein of GFP-C/EBP was applied to the columns and eluted with different salt concentrations. The active protein elutes in 0.5M NaCl and SDS-PAGE/silver stained gels show a single major band which comigrates with GFP-C/EBP as well as three minor contaminants. This provides a new alternative way of coupling DNA to solid supports using mild chemistry which is non-detrimental to the DNA and can be performed if required in the presence of nuclear extract.

A novel SP1/SP3 dependent intronic enhancer governing transcription of the UCP3 gene in brown adipocytes.

Uncoupling protein (UCP) 3 is a mitochondrial inner membrane protein implicated in lipid handling and metabolism of reactive oxygen species. Its transcription is mainly regulated by peroxisome proliferator-activated receptors (PPAR), a family of nuclear hormone receptors. Employing bandshift assays, RNA interference and reporter gene assays we examine an intronic region in the UCP3 gene harboring a cis-element essential for expression in brown adipocytes. We demonstrate binding of SP1 and SP3 to this element which is adjacent to a direct repeat 1 element mediating activation of UCP3 expression by PPARγ agonists. Transactivation mediated by these elements is interdependent and indispensable for UCP3 expression. Systematic deletion uncovered a third binding element, a putative NF1 site, in close proximity to the SP1/3 and PPARγ binding elements. Data mining demonstrated binding of MyoD and Myogenin to this third element in C2C12 cells, and, furthermore, revealed recruitment of p300. Taken together, this intronic region is the main enhancer driving UCP3 expression with SP1/3 and PPARγ as the core factors required for expression.

Asymmetric polymerase chain reaction provides alternatives for preparation of (GT)5-tailed duplex DNA promoter for promoter trapping.

Synthesis of (GT)5-tailed duplex DNA promoter is an important first step for purifying transcription complexes by promoter trapping purification. In our previous publication, we showed that the purification of the c-jun promoter using lambda exonuclease digestion of polymerase chain reaction (PCR) produced DNA with single-stranded tails. Asymmetric PCR can also produce tailed single strands that can be annealed to yield the desired promoter. An effective method uses asymmetric PCR and double digestion. After PCR, first a restriction enzyme, in this case SacII, cuts duplex strands remaining after asymmetric PCR, leaving 5' phosphoryl ends susceptible to a second digestion with lambda exonuclease to effectively degrade any duplex. The resulting single strands are then annealed to produce a duplex DNA with a single-stranded (GT)5 tail at the 3' end of each strand of the duplex. Unlike the previously described method, this novel procedure produces the desired tailed promoter devoid of any untailed duplex.

Laminin-α1 LG4-5 domain binding to dystroglycan mediates muscle cell survival, growth, and the AP-1 and NF-κB transcription factors but also has adverse effects.

In our previous studies, we showed laminin binds α-dystroglycan in the dystrophin glycoprotein complex and initiates cell signaling pathways. Here, differentiated C2C12 myocytes serve as a model of skeletal muscle. C2C12 cells have a biphasic response to the laminin-α(1) laminin globular (LG) 4-5 domains (1E3) dependent on the concentration used; at low concentrations of 1E3 (<1 µg/ml), myoblast proliferation is increased while higher concentrations (>1 µg/ml) cause apoptosis in myoblasts and differentiated myotubes. This alters the activation of the transcription factors activator protein-1 (AP-1) and NF-κB via laminin-dystrophin glycoprotein complex (DGC)-src-grb2-sos1-Rac1-Pak1-c-jun N-terminal kinase (JNK)p46 and laminin-DGC-Gßγ-phosphatidylinositol 3-kinase (PI3K)-Akt pathways, respectively. A specific antibody against Ser(63) phosphorylated c-jun completely blocks or supershifts the AP-1-DNA binding resulting from laminin binding but only partially blocks or supershifts the AP-1-DNA binding resulting from 1E3. This suggests that AP-1 contains phosphorylated c-jun in the presence of hololaminin but contains a different composition in the presence of 1E3. Nuclear NF-κB was only upregulated by a low concentration of 1E3 and is then diminished by a higher concentration; it also has a biphasic response. Nuclear localization of NF-κB is affected by PI3K/Akt signaling, and DGC associated PI3K activity also shows a biphasic response to 1E3. Furthermore, our data suggest that activation of c-jun N-terminal kinase participates in the cell survival pathway and suggest that NF-κB is involved in both survival and cell death. A model is presented which incorporates these observations.

Proteomic methodologies to study transcription factor function.

Transcription factors regulate transcription by binding to regulatory regions of genes including the promoter. Few of the transcription factors are well characterized, and few promoters have been described in detail. New methods have been developed to improve both transcription factor and promoter characterization, some of which are discussed here. Trapping methodology applicable to both individual transcription factors and intact transcription complexes are described, as well as 2D gel electrophoresis, Southwestern blotting, and basic liquid chromatography/tandem mass spectrometry methodology. These methods have proved useful in the study of transcriptional regulation.

CCR5 promoter haplotype transcription complex characterization.

CC chemokine receptor 5 (CCR5) is a major coreceptor for cell entry of human immunodeficiency virus (HIV); its expression is highly associated with virus replication and susceptibility. Single nucleotide polymorphisms (SNPs) in the CCR5 promoter play a critical role in CCR5 transcriptional regulation. HHA and HHE represent two contrasting haplotypes of CCR5 with only two base pair differences in the promoter. Identifying the transcription factors (TFs) that differentially bind to the polymorphic sites (the SNPs) in CCR5 haplotypes aids understanding HIV transmission/pathogenesis. Promoter trapping and two-dimensional southwestern blot analysis, to purify transcription complex and identify the differential TFs binding profile, is combined with HPLC-ESI-MS/MS, to determine those proteins specifically bound to one haplotype. This strategy reveals clear differences in haplotype-TF binding and has great promise for investigating how the CCR5 haplotypes may affect HIV-AIDS (acquired immune deficiency syndrome) susceptibility or disease progression.

Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis.

Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC-nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis (EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC-nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract, as well as purifying AP1 and CEBP DNA binding proteins from a human embryonic kidney cell line (HEK293) nuclear extract. AP1 components, c-Jun, Jun-D, c-Fos, CREB, ATF1 and ATF2 were successfully identified from 1.5 mg of nuclear extract (equivalent to 3×10(7) HEK293 cells) with AP1 binding activity of 750 fmol. In conclusion, this new strategy of combining EMSA with additional dimensions of electrophoresis and using southwestern blotting for detection proves to be a valuable approach in the identification of transcriptional complexes by proteomic methods.

Repeated probing of Southwestern blots using alkaline phosphatase stripping.

Southwestern blotting is when a DNA sequence is used to probe DNA-binding proteins on an electrophoretic gel blot. It would be highly desirable to be able to probe a blot repeatedly with different DNA sequences. Alkaline phosphatase can remove 5'-phosphoryl groups from DNA and radiolabeled 5'-(32)P-DNA probes are commonly used in Southwestern blotting. Here is shown that once probed, the radioisotope signal on the blot can be effectively removed by brief digestion with alkaline phosphatase, and the blot can then be repeatedly probed at least six times with different DNA probes. This exceeds the repetitions possible with another commonly used method using SDS. The technique can be used with either one-dimensional or multi-dimensional Southwestern blots and does not have a large effect on the phosphorylation state of the blotted proteins. An alternative method using T4 polynucleotide kinase stripping is also introduced but was less well characterized.

Purification and identification of a transcription factor, USF-2, binding to E-box element in the promoter of human telomerase reverse transcriptase (hTERT).

Controversy remains about the identity of the transcription factor(s) (TFs), which bind to the two E-box elements (CACGTG, proximal and distal) of the human telomerase (hTERT) gene promoter, the essential elements in the regulation of telomerase. Here, systematic oligonucleotide trapping supplemented with 2-DE and proteomic methods was used to identify E-box binding TFs. Although insufficient purity was obtained from the proximal E-box element trapping, further fractionation provided by 2-DE and specific identification from Southwestern blotting analysis allow us to clearly identify an E-box binding TF. The protein spot was cut from 2-DE and in-gel digested with trypsin for LC-nanospray ESI-MS/MS analysis. This identified upstream stimulatory factor 2 (USF2). Western blotting analysis with specific antibodies clearly shows USF2 present in the purified fraction and USF2 antibody supershifts the specific DNA-binding complex on non-denaturing gels. Furthermore, a novel method was developed in which the specific DNA-TF complex was separated on a non-denaturing gel, the band was cut and applied to SDS-PAGE for a second dimension. Western blots of this second gel also confirmed the presence of USF2.

LG4-5 domains of laminin-211 binds alpha-dystroglycan to allow myotube attachment and prevent anoikis.

Poly(2-hydroxyethyl methacrylate) (PolyHEMA) prevents cell attachment was used here to study anoikis, the process where cells die when unattached or attached to an inappropriate matrix, in mouse C(2)C(12) myotubes. A method was developed to efficiently embed proteins into PolyHEMA and the effect on cultured myotubes was determined. Myotubes grown on PolyHEMA-coated plates fail to attach to the surface and remain as rounded, suspended cells, undergo dramatic increases in apoptosis and necrosis, and the number of viable cells decreases. Incorporation of merosin (laminin-211) or the short laminin globular (LG4-5) modules of the laminin-alpha2 chain C-terminus (called 2E3) that binds alpha-dystroglycan diminishes both apoptosis and necrosis and increases viability while bovine serum albumin had a much lesser effect, showing the specificity of this effect for these matrix proteins. One sarcolemma receptor for laminin-binding is alpha-dystroglycan. An antibody which binds alpha-dystroglycan but which does not block laminin-binding (VIA4) had little effect on apoptosis or viability on merosin or 2E3 embedded plates while another antibody (IIH6) which specifically blocks binding dramatically decreased viability and increased apoptosis. When merosin or 2E3 are added to culture media rather than embedded on plates these can also increase viability and decrease apoptosis even though the cells remain in suspension, though the effect is not as great as found for the embedded proteins where the cells attach. Thus, we conclude that the binding of a small LG4-5 modules of laminin-211 to alpha-dystroglycan is important in preventing anoikis and that attachment plus binding is necessary for maximal cell survival.

Methods for proteomic analysis of transcription factors.

Investigation of the transcription factor (TF) proteome presents challenges including the large number of low abundance and post-translationally modified proteins involved. Specialized purification and analysis methods have been developed over the last decades which facilitate the study of the TF proteome and these are reviewed here. Generally applicable proteomics methods that have been successfully applied are also discussed. TFs are selectively purified by affinity techniques using the DNA response element (RE) as the basis for highly specific binding, and several agents have been discovered that either enhance binding or diminish non-specific binding. One such affinity method called "trapping" enables purification of TFs bound to nM concentrations and recovery of TF complexes in a highly purified state. The electrophoretic mobility shift assay (EMSA) is the most important assay of TFs because it provides both measures of the affinity and amount of the TF present. Southwestern (SW) blotting and DNA-protein crosslinking (DPC) allow in vitro estimates of DNA-binding-protein mass, while chromatin immunoprecipitation (ChIP) allows confirmation of promoter binding in vivo. Two-dimensional gel electrophoresis methods (2-DE), and 3-DE methods which combines EMSA with 2-DE, allow further resolution of TFs. The synergy of highly selective purification and analytical strategies has led to an explosion of knowledge about the TF proteome and the proteomes of other DNA- and RNA-binding proteins.

Two-dimensional southwestern blotting and characterization of transcription factors on-blot.

Two-dimensional Southwestern blotting (2D-SW) described here combines several steps. Proteins are separated by two-dimensional gel electrophoresis and transferred to nitrocellulose (NC) or polyvinylidene fluoride (PVDF) membrane. The blotted proteins are then partially renatured and probed with a specific radiolabeled oligonucleotide for Southwestern blotting (SW) analysis. The detected proteins are then processed by on-blot digestion and identified by LC-MS/MS analysis. A transcription factor, bound by a specific radiolabeled element, is thus characterized without aligning with protein spots on a gel. In this study, we systematically optimize conditions for 2D-SW and on-blot digestion. By quantifying the SW signal using a scintillation counter, the optimal conditions for SW were determined to be PVDF membrane, 0.5% PVP40 for membrane blocking, serial dilution of guanidine HCl for denaturing and renaturing proteins on the blot, and an SDS stripping buffer to remove radiation from the blot. By the quantification of the peptide yields using nano-ESI-MS analysis, the optimized conditions for on-blot digestions were found to be 0.5% Zwittergent 3-16 and 30% acetonitrile in trypsin digestion buffer. With the use of the optimized 2D-SW technique and on-blot digestion combined with HPLC-nano-ESI-MS/MS, a GFP-C/EBP model protein was successfully characterized from a bacterial extract, and native C/EBP beta was identified from 100 microg of HEK293 nuclear extract without any previous purification.