1776 SSLRGF(41)[1.087 ± 0.059] 1 Phage display (competitive panning) PMID:8623148 GSI-B4 isolectin Alpha D-Galactose 1HQL, Not determined. f3-6mer phage display library (X6) ELISA In phage ELISA, the absorbance at 505 nm was determined. Data were reproduced from the graph and shown as means ± standard deviations. Phage displaying peptide WAGSTR originated from the random library that had not been biopanned with the absorbance of 0.268 ± 0.028. A phage (displaying PARENT) from which the peptide library was generated is included as a control with the absorbance of 0.260 ± 0.036. The 100-mm tissue culture plates were coated with streptavidin prior to capture of biotinylated GS-1-B4. Phages bound to GS-1-B4 were eluted with melibiose obtained from Sigma. The plates thus prepared were then biopanned against the random peptide library. After biopanning, approximately 100 randomly picked clones were sequenced through the random peptide insertion site to identify potential motifs. Nearly 40% of the phage clones identified from GS-1-B4 biopanning contained peptide SSLRGF. This peptide blocks the binding of GS-1-B4 to pig aortic endothelial cells. The carbohydrate Gal alpha (1,3)Gal competes with the binding of GS-1-B4 to the peptide, suggesting that they may bind the same site. Using a RBC agglutination assay, we show that this peptide inhibits the agglutination of pig RBCs by heat-inactivated human serum at concentrations similar to that of Gal alpha (1,3)Gal. 1777 RNEPPL(3) LWYATE(3) SQMPPL(3) RGAIEP(2) FSAATE(2) ATESAM(1) ATEVVG(1) ATEARE(1) RLDPPI(1) RWDPPI(1) 10 Phage display (common panning) PMID:8623148 Anti-pig Xenoreactive Natural Antibodies, XNAs Gal alpha (1,3)Gal Not determined. Not determined. f3-6mer phage display library (X6) The 100-mm tissue culture plates were coated with streptavidin prior to capture of biotinylated XNAs. The plates thus prepared were then biopanned against the random peptide library. 1778 FGRLVSSIRY(1) TWKTSRISIF(6) 2 Phage display (subtractive panning, competitive panning) 5 PMID:9894906 Fc domain of IgG (IGHG1,IGHG2,IGHG3,IGHG4) Immunoglobulin G-binding protein A 1FC2, Not determined. X10 fUSE5 phage display library The decapeptide library was preselected against streptavidin-coated paramagnetic beads to reduce the number of phage clones interacting with streptavidin or with the plastic. The reduced library was then subjected to five selections against IgG Fc. In the first selection, interacting phage peptides were first eluted with protein A and then by lowering the pH. In subsequent selections, phages were elute by lowering the pH. Individual peptide phage clones were found to interact more strongly with IgG Fc than did either the original library or the wild-type phage. It was found that increasing concentrations of protein A competitively reduced the interaction of FARLVSSIRY-phage clone with IgG Fc to the same level as the primary library, indicating that this peptide really interacts with the protein A binding site on IgG Fc. On the other hand, it was only possible to reduce marginally the interaction of TWKTSRISIF-phage clone with IgG Fc using protein A as a competitor. When immunoglobulins from chicken, donkey, human, mouse, swine, rabbit, and sheep were included, peptide phage clones FGRLVSSIRY and TWKTSRISIF interacted strongly with human IgG Fc and porcine IgG and weakly with the immunoglobulins obtained from the other species. 1779 RLWLHRHKLV(1) FARLVSSIRY(1) FGRLVSSIRY(2) TWKTSRISIF(2) 4 Phage display (subtractive panning, competitive panning) 5 PMID:9894906 Fc domain of IgG (IGHG1,IGHG2,IGHG3,IGHG4) Immunoglobulin G-binding protein A 1FC2, Not determined. X10 fUSE5 phage display library The decapeptide library was preselected against streptavidin-coated paramagnetic beads to reduce the number of phage clones interacting with streptavidin or with the plastic. The reduced library was then subjected to five selections against IgG Fc. In the first selection, interacting phage peptides were first eluted with protein A and then by lowering the pH. In subsequent selections, phages were elute by protein A. It was found that increasing concentrations of protein A competitively reduced the interaction of FARLVSSIRY-phage clone with IgG Fc to the same level as the primary library, indicating that this peptide really interacts with the protein A binding site on IgG Fc. On the other hand, it was only possible to reduce marginally the interaction of TWKTSRISIF-phage clone with IgG Fc using protein A as a competitor. When immunoglobulins from chicken, donkey, human, mouse, swine, rabbit, and sheep were included, peptide phage clones FGRLVSSIRY and TWKTSRISIF interacted strongly with human IgG Fc and porcine IgG and weakly with the immunoglobulins obtained from the other species. 1780 EPMTPHQWITLYRSY(0.37) DTPYPWGWLLDEGYD(0.22) FCPPILPYSAWCPVP(0.10) MPVSRLCIELDWCPP(0.10) RGTQEWTELWVSFRA(0.05) RTGHSHDPRSMPKSC(0.05) YGTAPEWVLAFRLAF(0.02) RHRSGTASASKMSPI(0.02) VSPMEIWTTCTWWSS(0.02) SRGSHEWAVLFRFYY(0.02) 10 Phage display (subtractive panning, competitive panning) 4 PMID:10600340 IgG from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients Not determined. Not determined. Not determined. f88-15mer phage display library (X15) The cerebrospinal fluids (100 ml, about 5a??a Thirty-six clones were randomly picked and sequenced. The two peptides (DTPYPWGWLLDEGYD and RTGHSHDPRSMPKSC) were not specifically recognized by either cerebrospinal fluids from multiple sclerosis patient or from viral myelitis patient. In contrast, FCPPILPYSAWCPVP, EPMTPHQWITLYRSY, SRGSHEWAVLFRFYY, MPVSRLCIELDWCPP and RGTQEWTELWVSFRA were significantly recognized by cerebrospinal fluids from multiple sclerosis patient but not by cerebrospinal fluids from other neurological diseases. The combination of 4 selected mimotopes (FCPPILPYSAWCPVP, SRGSHEWAVLFRFYY, RGTQEWTELWVSFRA, EPMTPHQWITLYRSY), allowed the detection of specific antibodies in 21 of 60 multiple sclerosis cerebrospinal fluids whereas only 2 of 27 cerebrospinal fluids from patients with other neurological diseases equally recognized the 4 mimotopes. 1781 EPMTPHQWITLYRSY(0.66) SRGSHEWAVLFRFYY(0.09) FCPPILPYSAWCPVP(0.06) RGTQEWTELWVSFRA(0.06) QSPLEDRILRFLSPP(0.06) 5 Phage display (common panning) 3 PMID:10600340 IgG from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients Not determined. Not determined. Not determined. f88-15mer phage display library (X15) The cerebrospinal fluids from only one multiple sclerosis patient was used as the target, which was imobilized on to the streptavidin-coated polystyrene petri dishes through biotinylated mouse anti-human IgG monoclonal antibody P5F2F7. The three rounds of biopanning were performed using decreasing amounts of the same cerebrospinal fluids from the multiple sclerosis patient (10, 1, and 0.1 mg of IgG, respectively). Thirty-six clones were randomly picked and sequenced. FCPPILPYSAWCPVP, EPMTPHQWITLYRSY, SRGSHEWAVLFRFYY and RGTQEWTELWVSFRA were significantly recognized by cerebrospinal fluids from multiple sclerosis patient but not by cerebrospinal fluids from other neurological diseases. The combination of 4 selected mimotopes (FCPPILPYSAWCPVP, SRGSHEWAVLFRFYY, RGTQEWTELWVSFRA, EPMTPHQWITLYRSY), allowed the detection of specific antibodies in 21 of 60 multiple sclerosis cerebrospinal fluids whereas only 2 of 27 cerebrospinal fluids from patients with other neurological diseases equally recognized the 4 mimotopes. 1782 EPMTPHQWITLYRSY(0.30) FCPPILPYSAWCPVP(0.15) MPVSRLCIELDWCPP(0.11) QSPLEDRILRFLSPP(0.07) SSRQGLIDCLWSCTH(0.07) SRGSHEWAVLFRFYY(0.04) VSPMEIWTTCTWWSS(0.04) 7 Phage display (common panning) 3 PMID:10600340 IgG from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients Not determined. Not determined. Not determined. f88-15mer phage display library (X15) The cerebrospinal fluids from only one multiple sclerosis patient was used as the target, which was imobilized on to the streptavidin-coated polystyrene petri dishes through biotinylated mouse anti-human IgG monoclonal antibody P5F2F7. The three rounds of biopanning were performed using decreasing amounts of the same cerebrospinal fluids from the multiple sclerosis patient (10, 1, and 0.1 mg of IgG, respectively). Thirty-six clones were randomly picked and sequenced. FCPPILPYSAWCPVP, EPMTPHQWITLYRSY, SRGSHEWAVLFRFYY and MPVSRLCIELDWCPP were significantly recognized by cerebrospinal fluids from multiple sclerosis patient but not by cerebrospinal fluids from other neurological diseases. The combination of 4 selected mimotopes (FCPPILPYSAWCPVP, SRGSHEWAVLFRFYY, RGTQEWTELWVSFRA, EPMTPHQWITLYRSY), allowed the detection of specific antibodies in 21 of 60 multiple sclerosis cerebrospinal fluids whereas only 2 of 27 cerebrospinal fluids from patients with other neurological diseases equally recognized the 4 mimotopes. 1783 FCPPILPYSAWCPVP(0.26) MPVSRLCIELDWCPP(0.23) EPMTPHQWITLYRSY(0.20) QSPLEDRILRFLSPP(0.06) SRGSHEWAVLFRFYY(0.03) VSPMEIWTTCTWWSS(0.03) SSRQGLIDCLWSCTH(0.03) 7 Phage display (common panning) 3 PMID:10600340 IgG from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients Not determined. Not determined. Not determined. f88-15mer phage display library (X15) The cerebrospinal fluids from only one multiple sclerosis patient was used as the target, which was imobilized on to the streptavidin-coated polystyrene petri dishes through biotinylated mouse anti-human IgG monoclonal antibody P5F2F7. The three rounds of biopanning were performed using decreasing amounts of the same cerebrospinal fluids from the multiple sclerosis patient (10, 1, and 0.1 mg of IgG, respectively). Thirty-six clones were randomly picked and sequenced. FCPPILPYSAWCPVP, EPMTPHQWITLYRSY, SRGSHEWAVLFRFYY and MPVSRLCIELDWCPP were significantly recognized by cerebrospinal fluids from multiple sclerosis patient but not by cerebrospinal fluids from other neurological diseases. The combination of 4 selected mimotopes (FCPPILPYSAWCPVP, SRGSHEWAVLFRFYY, RGTQEWTELWVSFRA, EPMTPHQWITLYRSY), allowed the detection of specific antibodies in 21 of 60 multiple sclerosis cerebrospinal fluids whereas only 2 of 27 cerebrospinal fluids from patients with other neurological diseases equally recognized the 4 mimotopes. 1784 NACYVDLFLGASVCP(0.46)[0.07] SSAKSHCYAFCSGLP(0.31)[0.25] HCRKVTGSDYLLCGL(0.05)[0.05] 3 Phage display (subtractive panning, competitive panning) 4 PMID:10600340 IgG from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients Not determined. Not determined. Not determined. f88-15mer phage display library (X15) ELISA IgG immunoreactivity of MS sera against the selected phage-displayed peptides was detected by ELISA on immobilized phages with a pool of five MS sera diluted to 1:100. The results are expressed as the difference between the OD obtained at 492 nm with MS sera and that with non-MS sera. The serum (20 ml, about 200 mg of IgG) from multiple sclerosis patients was used as the target, which was imobilized on to the streptavidin-coated polystyrene petri dishes through biotinylated mouse anti-human IgG monoclonal antibody P5F2F7. For each round of panning, serum from a different multiple sclerosis patient was used. In the fourth round, the amplified eluate was incubated with 100 ml of a pool of sera from five non-multiple sclerosis patients before reacting with dish-bound serum IgG from multiple sclerosis patient. Thirty-six clones were randomly picked and sequenced. Compared to control peptide, NACYVDLFLGASVCP and HCRKVTGSDYLLCGL were not specifically recognized by individual multiple sclerosis sera whereas SSAKSHCYAFCSGLP was significantly recognized by only 3 of 37 sera. 1785 NACYVDLFLGASVCP(0.50)[0.07] HCRKVTGSDYLLCGL(0.33)[0.05] SSAKSHCYAFCSGLP(0.06)[0.25] SPLNLCFERCFTSPN(0.06)[ND] SHPTLMPPLARMPSL(0.03)[ND] 5 Phage display (subtractive panning, competitive panning) 4 PMID:10600340 IgG from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients Not determined. Not determined. Not determined. f88-15mer phage display library (X15) ELISA IgG immunoreactivity of MS sera against the selected phage-displayed peptides was detected by ELISA on immobilized phages with a pool of five MS sera diluted to 1:100. The results are expressed as the difference between the OD obtained at 492 nm with MS sera and that with non-MS sera. The serum (20 ml, about 200 mg of IgG) from multiple sclerosis patients was used as the target, which was imobilized on to the streptavidin-coated polystyrene petri dishes through biotinylated mouse anti-human IgG monoclonal antibody P5F2F7. For each round of panning, serum from a different multiple sclerosis patient was used. In the fourth round, the amplified eluate was incubated with 100 ml of a pool of sera from five non-multiple sclerosis patients before reacting with dish-bound serum IgG from multiple sclerosis patient. Thirty-six clones were randomly picked and sequenced. Compared to control peptide, NACYVDLFLGASVCP and HCRKVTGSDYLLCGL were not specifically recognized by individual multiple sclerosis sera whereas SSAKSHCYAFCSGLP was significantly recognized by only 3 of 37 sera. 1786 AGPDCSSLPNSMRCS(6) GSRPSPIGSKWALQP(2) TSPHRPSAIGVLPPL(2) EKERRPSPIGTATLL(1) GKRPSCTGCSYELAQ(1) GGRHPSPMGTFDTQT(1) STKPSSIGTLHTSPS(1) AVSGIGTQTSIPYPS(1) LTTEPDTTKFWPAPS(1) SPPLQCSQYPNSLRC(1) QQPVSSWVFVSSDAI(1) SRPSP 11 Phage display (subtractive panning) 5 PMID:10848928 Anti-IgE polyclonal antibody IgG Ig epsilon chain C region Not determined. Not determined. f88-15mer phage display library (X15) Ten μg of biotinylated goat antihuman IgG (capture antibody) incubated with 1 mg of streptavidin-coated Dynabeads M-280. The beads were then washed five times in Tris-buffered saline (TBS) pH 7.4 and resuspended in 100mL TBS. This procedure was repeated with a second aliquot (1 mg) of beads. Ten mg of human IgG anti-IgE (selector antibody) was incubated with the first aliquot of beads for 2 hours at room temperature, and the beads were then washed four times in TBS. From the amplified phage-peptide library 60μL was added to the control beads and the mixture was incubated for 3 h at room temperature. This was done to remove any phage-peptides binding to the biotinylated antihuman IgG. The phage-peptide library, treated in this way, was then incubated with the IgG anti-IgE coated beads for 3 h at room temperature. 1787 VAWCTIFLCLDV[0.22] ADFCEGKDMIDWVYCRLY[2.5] FWFCDRIAWYPQHLCEFL[1.9] FRNCEPWMLRFGCNPR[4.8] 4 Phage display (common panning) 2 PMID:11934284 Human serum albumin Not determined. Not determined. Not determined. TN phage display library pool Fluorescence anisotropy Fluorescence anisotropy measurements were performed in various conditions. It was discovered that many of the peptides bound better at a lower pH (3 mM phosphate, pH 6.2) and in the absence of added salt. The dissociation constants (KD, μM) of this condition were shown. The pooled phage libraries of TN6-6, TN10-9, and TN12-1 were used in selections against human serum albumin (HSA) immobilized directly on polystyrene microtiter plates. After two rounds of selection, phage peptide selection on directly immobilized HSA yielded several phage isolates (232, 234, 236, and 238) that showed positive HSA binding by ELISA. Phage isolate 232 (VAWCTIFLCLDV) showed the highest ELISA signal. Although in solution DX-232 bound HSA with the highest affinity, the DX-232-Sepharose column performance was bad, binding no detectable HSA. In contrast, the DX-236(FWFCDRIAWYPQHLCEFL)-Sepharose column was the best performer and quantitatively bound the entire 1 mg injection (0.15 μmol). At higher HSA loads in the no salt, pH 6.2 buffer, the DX-236-Sepharose column (0.35 mL) bound upward of 4 mg of HSA, which corresponds to a dynamic capacity of greater than 11 mg/mL. The DX-236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. DX-236 also bound well to several mammalian serum albumins. 1788 FKICDQWFCLMP[1.8] HVGCNNALCMQY[17] WKVCDHFFCLSP[18] NHGCWHFSCIWD[1.9] 4 Phage display (subtractive panning, competitive panning) 4 PMID:11934284 Human serum albumin Not determined. Not determined. Not determined. TN6-6 phage display library Fluorescence anisotropy Fluorescence anisotropy measurements were performed in various conditions. It was discovered that many of the peptides bound better at a lower pH (3 mM phosphate, pH 6.2) and in the absence of added salt. The dissociation constants (KD, μM) of this condition were shown. The TN6-6 phage library was processed to remove bead binders and then screened against caprylate-biotinylated-HSA in solution for 1h; later on, HSA-binding phage were captured by the addition of magnetic streptavidin beads for 15 min. Phage isolates 298 (NHGCWHFSCIWD) showed the highest ELISA signal. 1789 DWDCVTRWANRDQQCWGP[9.5] DWDCVTRWANRDQQCWAL[13] DWDCVTDWANRHQHCWAL[6.7] DWQCVKDWANRRRGCMAD[17] RNMCKFSWIRSPAFCARA[0.9] 5 Phage display (subtractive panning, competitive panning) 4 PMID:11934284 Human serum albumin Not determined. Not determined. Not determined. TN12-1 phage display library Fluorescence anisotropy Fluorescence anisotropy measurements were performed in various conditions. It was discovered that many of the peptides bound better at a lower pH (3 mM phosphate, pH 6.2) and in the absence of added salt. The dissociation constants (KD, μM) of this condition were shown. The TN12-1 phage library was processed to remove bead binders and then screened against caprylate-biotinylated-HSA in solution for 1h; later on, HSA-binding phage were captured by the addition of magnetic streptavidin beads for 15 min. Phage isolates 321 (RNMCKFSWIRSPAFCARA) showed the highest ELISA signal. DX-321 had a KD value less than 2μM in no salt buffer at pH 6.2. It also showed a strong preference for albumin from humans. 1790 NCVSPYWCEPLAPSARA 1 Phage display (competitive panning) PMID:12890473 GSI-B4 isolectin Alpha D-Galactose 1HQL, Not determined. XCX15 phage display library Phages bound to GS-1-B4 were eluted with melibiose obtained from Sigma. After biopanning, 18 clones were randomly picked. On four out of them was performed the ELISA to compare the binding activity. All clones selected were able to bind the lectin BS-I-B4. A phage bearing the peptide NCVSPYWCEPLAPSARA has been identified to bind the lectin strongly. Melibiose was able to inhibit the binding of the human natural anti-alpha-Gal antibody to the peptide competitively. The peptide also inhibited the agglutination of pig RBCs caused by human natural antibody or lectin BS-I-B4. 1791 HLEPLIS(10) 1 Phage display (common panning) 1 PMID:15472891 Streptavidin Biotin 1DF8,1LUQ,1MEP,1MK5,1N43,1N9M,1NDJ,1NQM,1STP,1SWD,1SWE,1SWG,1SWK,1SWN,1SWP,1SWR,1SWT,2F01,2GH7,2IZF,2IZG,2IZH,2IZI,2IZJ,2RTD,2RTE,2RTF,2RTG,2Y3F,3MG5, Not determined. Ph.D.-7 phage display library (X7) In total, 10 clones were sequenced. 1792 HLEPLIS(6) 1 Phage display (common panning) 2 PMID:15472891 Streptavidin Biotin 1DF8,1LUQ,1MEP,1MK5,1N43,1N9M,1NDJ,1NQM,1STP,1SWD,1SWE,1SWG,1SWK,1SWN,1SWP,1SWR,1SWT,2F01,2GH7,2IZF,2IZG,2IZH,2IZI,2IZJ,2RTD,2RTE,2RTF,2RTG,2Y3F,3MG5, Not determined. Ph.D.-7 phage display library (X7) In total, 6 clones were sequenced. 1793 TESMLPS(2) 1 Phage display (competitive panning) 1 PMID:15472891 SB-365579 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) All incubation steps were carried out at room temperature. NeutrAvidin-coated plates were used to capture SB-365579. To reduce nonspecific binding, a blocking step was performed using 5% skim milk. All of the compounds were initially dissolved in dimethyl formamide (DMF) (22 mM drug stock solutions) and then diluted in TBSTM (tris-buffered saline, pH 7.5, with 0.1% Tween-20 and 5% skim milk) to a final concentration of 22 mM. Bound phage from the drug-coated wells were eluted with 200 μl of 220 μM SB-365579 solution in TBSTM. After the fisrt round of panning, 29 clones were sequenced. However, many sequences were not given in the original paper. 1794 HLEPLIS(6) ATLAPFT(2) 2 Phage display (competitive panning) 2 PMID:15472891 SB-365579 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) All incubation steps were carried out at room temperature. NeutrAvidin-coated plates were used to capture SB-365579. To reduce nonspecific binding, a blocking step was performed using 5% skim milk. All of the compounds were initially dissolved in dimethyl formamide (DMF) (22 mM drug stock solutions) and then diluted in TBSTM (tris-buffered saline, pH 7.5, with 0.1% Tween-20 and 5% skim milk) to a final concentration of 22 mM. Bound phage from the drug-coated wells were eluted with 200 μl of 220 μM SB-365579 solution in TBSTM. After the second round of panning, 21 clones were sequenced. However, many sequences were not given in the original paper. 1795 LPLTPLP(8) ATLAPFT(6) HLEPLIS(2) 3 Phage display (competitive panning) 3 PMID:15472891 SB-365579 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) All incubation steps were carried out at room temperature. NeutrAvidin-coated plates were used to capture SB-365579. To reduce nonspecific binding, a blocking step was performed using 5% skim milk. All of the compounds were initially dissolved in dimethyl formamide (DMF) (22 mM drug stock solutions) and then diluted in TBSTM (tris-buffered saline, pH 7.5, with 0.1% Tween-20 and 5% skim milk) to a final concentration of 22 mM. Bound phage from the drug-coated wells were eluted with 200 μl of 220 μM SB-365579 solution in TBSTM. After the third round of panning, 26 clones were sequenced. However, many sequences were not given in the original paper. 1796 TPSPERP(12) LPLTPLP(7) ATLAPFT(6) ATPLWLK(1) 4 Phage display (competitive panning) 1 PMID:15472891 SB-365580 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) All incubation steps were carried out at room temperature. NeutrAvidin-coated plates were used to capture SB-365580. To reduce nonspecific binding, a blocking step was performed using 5% skim milk. All of the compounds were initially dissolved in dimethyl formamide (DMF) (22 mM drug stock solutions) and then diluted in TBSTM (tris-buffered saline, pH 7.5, with 0.1% Tween-20 and 5% skim milk) to a final concentration of 22 mM. Bound phage from the drug-coated wells were eluted with 200 μl of 220 μM SB-365580 solution in TBSTM. After the fisrt round of panning, 27 clones were sequenced. However, there was one sequence not given in the original paper. 1797 TPSPERP(5) LPLTPLP(8) ATLAPFT(3) ATPLWLK(1) 4 Phage display (competitive panning) 2 PMID:15472891 SB-365580 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) All incubation steps were carried out at room temperature. NeutrAvidin-coated plates were used to capture SB-365580. To reduce nonspecific binding, a blocking step was performed using 5% skim milk. All of the compounds were initially dissolved in dimethyl formamide (DMF) (22 mM drug stock solutions) and then diluted in TBSTM (tris-buffered saline, pH 7.5, with 0.1% Tween-20 and 5% skim milk) to a final concentration of 22 mM. Bound phage from the drug-coated wells were eluted with 200 μl of 220 μM SB-365580 solution in TBSTM. After the second round of panning, 17 clones were sequenced. 1798 TPSPERP(9) LPLTPLP(8) ATLAPFT(8) 3 Phage display (competitive panning) 3 PMID:15472891 SB-365580 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) All incubation steps were carried out at room temperature. NeutrAvidin-coated plates were used to capture SB-365580. To reduce nonspecific binding, a blocking step was performed using 5% skim milk. All of the compounds were initially dissolved in dimethyl formamide (DMF) (22 mM drug stock solutions) and then diluted in TBSTM (tris-buffered saline, pH 7.5, with 0.1% Tween-20 and 5% skim milk) to a final concentration of 22 mM. Bound phage from the drug-coated wells were eluted with 200 μl of 220 μM SB-365580 solution in TBSTM. After the third round of panning, 27 clones were sequenced. However, some sequences were not given in the original paper. 1799 KVWVLPI(51) 1 Phage display (competitive panning) 3 PMID:15574836 70 kDa heat shock cognate protein 1 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The binding ability of the high-frequency peptides was confirmed by ELISA. Absorbances at 405 nm were measured (data not shown). Normalized data were used to quantify the results. The heptapeptide KVWVLPI had the highest binding ability to the target protein Vr130. The recombinant proteins were designated Vr130 for polypeptides containing the C-terminal 30 kDa domain of VrHsc70-1. The recombinant proteins were purified on a metal-affinity column and then cleaved with thrombin to remove the His tag. The Vr130 recombinant proteins was used to select VrHsc70-binding heptapeptides using phage display. The bound phage was eluted with a 1 ml solution of the free target protein. Among 125 different clones selected with Vr130, the heptapeptide KVWVLPI had the highest frequency(40.8%). The binding ability of the high-frequency peptides was confirmed by ELISA. 1800 KLWVIPQ(54) 1 Phage display (competitive panning) 3 PMID:15574836 70 kDa heat shock cognate protein 1 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The binding ability of the high-frequency peptides was confirmed by ELISA. Absorbances at 405 nm were measured (data not shown). Normalized data were used to quantify the results. The peptide KLWVIPQ bound strongly to Vr230, but exhibited <5% binding ability towards Vr130. Conversely, KVWVLPI exhibited 13% binding ability towards Vr230. The recombinant proteins were designated Vr230 for polypeptides containing the C-terminal 30 kDa domain of VrHsc70-2. The recombinant proteins were purified on a metal-affinity column and then cleaved with thrombin to remove the His tag. The Vr230 recombinant proteins was used to select VrHsc70-binding heptapeptides using phage display. The bound phage was eluted with a 1 ml solution of the free target protein. Among 123 different clones of Vr230, the heptapeptide KLWVIPQ had the highest frequency (43.9%). The binding ability of the high-frequency peptides was confirmed by ELISA.