(B) Quantitative ELISA was performed with either immobilized FGFR1\Fc or Fc, to determine peptide specificity towards FGFR1 (C) Competitive ELISA detected decreased F8 clone binding to FGFR1 in the presence of FGF1, suggesting the possibility of overlapping binding interface. at 37?C and purified on a heparinCSepharose CL\6B resin according to described protocols 23, 24. Phage display biopanning A 96\well plate was coated with 100?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6) and then blocked with BSA for 2?h at 4?C. Afterwards the wells were washed five occasions with TBST (0.1% Tween\20 in Tris\buffered saline). The original library was diluted in TBST (final concentration 2??1011 plaque forming models, PFU) and 100?L was added to each coated well for 2?h at 4?C with gentle agitation. After washing the plate 10 occasions with TBST, the bounded phages were eluted with 0.2?m glycine/HCl (pH 2.2) and neutralized with 1?m Tris/HCl (pH 9.1). The eluted phages were then amplified, precipitated with polyethylene glycol/NaCl and titrated according to standard protocol (NEB). In the second round of selection there was an additional counterselection step for the Fc fragment. Fc at a concentration of 50?gmL?1 was immobilized in wells and blocked with 5% non\fat milk. Subsequently, the phages (2??1011?PFU) were incubated with Fc for 1?h at 4?C. Afterwards, phage clones were transferred to wells with FGFR1\Fc (70?gmL?1) and incubated for 1?h at 4?C with gentle agitation. After each round of selection, amplified phages were diluted and utilized for the next round of biopanning. In the third round of selection, the concentration of Rabbit Polyclonal to TIE2 (phospho-Tyr992) immobilized FGFR1\Fc and Fc was decreased down to 50 and 20?gmL?1, respectively. Additionally after the last round of selection, binding phages were eluted with 100 molar excess of FGF1 over applied phage library (2??1011?PFU). ELISA screening ELISA was conducted after the third round of selection. The 96\well Maxisorp F plate was coated with FGFR1\Fc (5?g per well), incubated at 4?C overnight and additionally blocked with 3% BSA for 2?h at 4?C. After washing with TBST (0.2% Tween\20) inoculated phage clones were added to the wells and incubated for 2?h at 4?C. Afterward the plate was washed five occasions and horseradish peroxidase (HRP)Canti\M13\monoclonal antibody (mAB) (1?:?5000?v/v, no. 27\9421\01; GE Healthcare, Chicago, IL, USA) was added to each well and incubated at room heat (RT) for 1?h. Subsequently, the plate was washed four occasions and 3,3,5,5\tetramethylbenzidine (TMB) was utilized for detection with the absorbance measured at 450?nm. Similarly, for the estimation of the level of Fc\binding, the plate was coated with Fc (5?g per well) and treated with phage clones as above. Quantitative ELISA The 96\well plate was coated with 50?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6), washed three times with PBS and subsequently blocked with 3% BSA for 2?h at 4?C. Subsequently, the wells were washed five occasions with PBS and 100?L of F8 and G10 phage clones (109?PFU) was added for 1?h at RT with gentle agitation. Afterwards the plate was washed five occasions with PBS and the HRPCanti\M13\mAB (1?:?5000?v/v) was added and incubated at RT for 1?h. Next, the plate was washed 10 occasions with PBS and substrate TMB was utilized for detection with the absorbance measured at 450?nm. Measurements were performed three times, each time in triplicate. Competitive ELISA An ELISA plate was coated with FGFR1\Fc as for quantitative ELISA. The wells were washed five occasions with PBS and 100?L of FGF1 (10?gmL?1) and phage clone (109?PFU) mix or phage alone was added for 1?h at 4?C with gentle agitation. After incubation the plate was washed five occasions with PBS and the detection with HRPCanti\M13\mAB (1?:?5000?v/v) and TMB substrate was performed as before. Measurements were performed three times, each time in triplicate. Peptide synthesis and disulfide bridge formation Peptides were synthesized manually with C\terminal amidation on Fmoc\solid phase according to standard strategy. The purity of obtained products was verified using RP\HPLC and the proper molar masses of synthesized peptides were confirmed by mass spectrometry. Disulfide bridge formation was optimized and oxidation folding in redox buffer protocol proved to be the.Normally the binder can be used as a therapeutic targeting agent only if a cytotoxic or diagnostic payload is attached to it. by over 40%. Such an effect was not observed for BA/F3 cells lacking FGFR1. Therefore, cyclic peptide F8 can act as a FGF1CFGFR1 conversation antagonist, and may be suitable for further development for potential use in therapies against FGFR1\expressing malignancy cells. BL21(DE3)pLysS strain at 37?C and purified on a heparinCSepharose CL\6B resin according to described protocols 23, 24. Phage display biopanning A 96\well plate was coated with 100?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6) and then blocked with BSA for 2?h at 4?C. Afterwards the wells were washed five occasions with TBST (0.1% Tween\20 in Tris\buffered saline). The original library was diluted in TBST (final concentration 2??1011 plaque forming models, PFU) and 100?L was added to each coated well for 2?h at 4?C with gentle agitation. After washing the plate 10 occasions with TBST, the bounded phages were eluted with 0.2?m glycine/HCl (pH 2.2) and neutralized with 1?m Tris/HCl (pH 9.1). The eluted phages were then amplified, precipitated with polyethylene glycol/NaCl and titrated according to standard protocol (NEB). In the second round of selection there was an additional counterselection step for the Fc fragment. Fc at a concentration of 50?gmL?1 was immobilized in wells and blocked with 5% non\fat milk. Subsequently, the phages (2??1011?PFU) were incubated with Fc for 1?h at 4?C. Afterwards, phage clones were transferred to wells with FGFR1\Fc (70?gmL?1) and incubated for 1?h at 4?C with gentle agitation. After each round of selection, amplified phages were diluted and utilized for the next round of biopanning. In the third round of selection, the concentration of immobilized FGFR1\Fc and Fc was decreased down to 50 and 20?gmL?1, respectively. Additionally after the last round of selection, binding phages were eluted with 100 molar excess of FGF1 over applied phage library (2??1011?PFU). ELISA screening ELISA was conducted after the third round of selection. The 96\well Maxisorp F plate was coated with FGFR1\Fc (5?g per well), incubated at 4?C overnight and additionally blocked with 3% BSA for 2?h at 4?C. After washing with TBST (0.2% Tween\20) inoculated phage clones were added to the wells and incubated for 2?h at 4?C. Afterward the plate was washed five times and horseradish peroxidase (HRP)Canti\M13\monoclonal antibody (mAB) (1?:?5000?v/v, no. 27\9421\01; GE Healthcare, Chicago, IL, USA) was added to each well and incubated at room temperature (RT) for 1?h. Subsequently, the plate was washed four times and 3,3,5,5\tetramethylbenzidine (TMB) was used for detection with the absorbance measured at 450?nm. Similarly, for the estimation of the level of Fc\binding, the plate was coated with Fc (5?g per well) and treated with phage clones as above. Quantitative ELISA The 96\well plate was coated with 50?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6), washed three times with PBS and subsequently blocked with 3% BSA for 2?h at 4?C. Subsequently, the wells were washed five times with PBS and 100?L of F8 and G10 phage clones (109?PFU) was added for 1?h at RT with gentle agitation. Afterwards the plate was washed five times with PBS and the HRPCanti\M13\mAB (1?:?5000?v/v) was added and incubated at RT for 1?h. Next, the plate was washed 10 times with PBS and substrate TMB was used for detection with the absorbance measured at 450?nm. Measurements were performed three times, each time in triplicate. Competitive ELISA An ELISA plate was coated with FGFR1\Fc as for quantitative ELISA. The wells were washed five times with PBS and 100?L of FGF1 (10?gmL?1) and phage clone (109?PFU) mix or phage alone was added for 1?h at 4?C with gentle agitation. After incubation the plate was washed five times with PBS and the detection with HRPCanti\M13\mAB (1?:?5000?v/v) and TMB substrate was performed as before. Measurements were performed three times, each time in triplicate. Peptide synthesis and disulfide bridge formation Peptides were synthesized manually with C\terminal amidation on Fmoc\solid phase according to standard strategy. The purity of obtained products was verified using RP\HPLC and the proper molar masses of synthesized peptides were confirmed by mass spectrometry. Disulfide bridge formation was optimized and oxidation folding in redox buffer protocol proved to be the most efficient method. Peptides at a final concentration of 0.1?mgmL?1 were dissolved in Milli\Q water (Merck Millipore; Burlington, MA, USA) at pH 3 and mixed with 4?m urea, 300?m reduced glutathione and 150?m oxidized glutathione. The pH was then adjusted to 8.7 with 1?m Tris/HCl and the solution was left overnight at RT with gentle stirring. Finally, cyclic peptides were purified via RP\HPLC and intramolecular disulfide bond formation was confirmed with matrix\assisted laser desorption/ionization time\of\flight mass spectrometry..Sequence similarity searches did not reveal any direct resemblance of selected peptides to either FGF family ligands or FGFR receptors. at 37?C and purified on a heparinCSepharose CL\6B resin according to described protocols 23, 24. Phage display biopanning A 96\well plate was coated with 100?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6) and then blocked with BSA for 2?h at 4?C. Afterwards the wells were washed five times with TBST (0.1% Tween\20 in Tris\buffered saline). The original library was diluted in TBST (final concentration 2??1011 plaque forming units, PFU) and 100?L was added to each coated well for 2?h at 4?C with gentle agitation. After washing the plate 10 times with TBST, the bounded phages were eluted with 0.2?m glycine/HCl (pH 2.2) and neutralized with 1?m Tris/HCl (pH 9.1). The eluted phages were then amplified, precipitated with polyethylene glycol/NaCl and titrated according to standard protocol (NEB). In the second round of selection there was an additional counterselection step for the Fc fragment. Fc at a concentration of 50?gmL?1 was immobilized in wells and blocked with 5% non\fat milk. Subsequently, the phages (2??1011?PFU) were incubated with Fc for 1?h at 4?C. Afterwards, phage clones were transferred to wells with FGFR1\Fc (70?gmL?1) and incubated for 1?h at 4?C with gentle agitation. After each round of selection, amplified phages were diluted and used for the next round of biopanning. In the third round of selection, the concentration of immobilized FGFR1\Fc and Fc was decreased down to 50 and 20?gmL?1, respectively. Additionally after the last round of selection, binding phages were eluted with 100 molar excess of FGF1 over applied phage library (2??1011?PFU). ELISA testing ELISA was carried out after the third round of selection. The 96\well Maxisorp F plate was coated with FGFR1\Fc (5?g per well), incubated at 4?C overnight and additionally blocked with 3% BSA for 2?h at 4?C. After washing with TBST (0.2% Tween\20) inoculated phage clones were added to the wells and incubated for 2?h at 4?C. Afterward the plate was washed five instances and horseradish peroxidase (HRP)Canti\M13\monoclonal antibody (mAB) (1?:?5000?v/v, no. 27\9421\01; GE Healthcare, Chicago, IL, USA) was added to each well and incubated at space temp (RT) for 1?h. Subsequently, the plate was washed four instances and 3,3,5,5\tetramethylbenzidine (TMB) was utilized for detection with the absorbance measured at 450?nm. Similarly, for the estimation of the level of Fc\binding, the plate was coated with Fc (5?g per well) and treated with phage clones while above. Quantitative ELISA The 96\well plate was coated with 50?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6), washed three times with PBS and subsequently blocked with 3% BSA for 2?h at 4?C. Subsequently, the wells were washed five instances with PBS and 100?L of F8 and G10 phage clones (109?PFU) was added for 1?h at RT with gentle agitation. Later on the plate was washed five instances with PBS and the Bitopertin (R enantiomer) HRPCanti\M13\mAB (1?:?5000?v/v) was added and incubated at RT for 1?h. Next, the plate was washed 10 instances with PBS and substrate TMB was utilized for detection with the absorbance measured at 450?nm. Measurements were performed three times, each time in triplicate. Competitive ELISA An ELISA plate was coated with FGFR1\Fc as for quantitative ELISA. The wells were washed five instances with PBS and 100?L of FGF1 (10?gmL?1) and phage clone (109?PFU) mix or phage alone was added for 1?h at 4?C with gentle agitation. After incubation the plate was washed five instances with PBS and the detection with HRPCanti\M13\mAB (1?:?5000?v/v) and TMB substrate was performed while before. Measurements were performed.In 2002, Roeske and colleagues identified a 6\amino\acid linear peptide targeting FGFR1. preventing the FGF1CFGFR1 connection, and also decreases FGF1\induced proliferation of BA/F3 FGFR1c cells by over 40%. Such an effect was not observed for BA/F3 cells lacking FGFR1. Consequently, cyclic peptide F8 can act as a FGF1CFGFR1 connection antagonist, and may be suitable for further development for potential use in therapies against FGFR1\expressing malignancy cells. BL21(DE3)pLysS strain at 37?C and purified Bitopertin (R enantiomer) on a heparinCSepharose CL\6B resin according to described protocols 23, 24. Phage display biopanning A 96\well plate was coated with 100?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6) and then blocked with BSA for 2?h at 4?C. Later on the wells were washed five instances with TBST (0.1% Tween\20 in Tris\buffered saline). The original library was diluted in TBST (final concentration 2??1011 plaque forming devices, PFU) and 100?L was added to each coated well for 2?h at 4?C with gentle agitation. After washing the plate 10 instances with TBST, the bounded phages were eluted with 0.2?m glycine/HCl (pH 2.2) and neutralized with 1?m Tris/HCl (pH 9.1). The eluted phages were then amplified, precipitated with polyethylene glycol/NaCl and titrated relating to standard protocol (NEB). In the second round of selection there was an additional counterselection step for the Fc fragment. Fc at a concentration of 50?gmL?1 was immobilized in wells and blocked with 5% non\fat milk. Subsequently, the phages (2??1011?PFU) were incubated with Fc for 1?h at 4?C. Later on, phage clones were transferred to wells with FGFR1\Fc (70?gmL?1) and incubated for 1?h at 4?C with gentle agitation. After each round of selection, amplified phages were diluted and utilized for the next round of biopanning. In the third round of selection, the concentration of immobilized FGFR1\Fc and Fc was decreased down to 50 and 20?gmL?1, respectively. Additionally after the last round of selection, binding phages were eluted with 100 molar excess of FGF1 over applied phage library (2??1011?PFU). ELISA testing ELISA was carried out after the third round of selection. The 96\well Maxisorp F plate was coated with FGFR1\Fc (5?g per well), incubated at 4?C overnight and additionally blocked with 3% BSA for 2?h at 4?C. After washing with TBST (0.2% Tween\20) inoculated phage clones were added to the wells and incubated for 2?h at 4?C. Afterward the plate was washed five occasions and horseradish peroxidase (HRP)Canti\M13\monoclonal antibody (mAB) (1?:?5000?v/v, no. 27\9421\01; GE Healthcare, Chicago, IL, USA) was added to each well and incubated at space heat (RT) for 1?h. Subsequently, the plate was washed four occasions and 3,3,5,5\tetramethylbenzidine (TMB) was utilized for detection with the absorbance measured at 450?nm. Similarly, for the estimation of the level of Fc\binding, the plate was coated with Fc (5?g per well) and treated with phage clones while above. Quantitative ELISA The 96\well plate was coated with 50?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6), washed three times with PBS and subsequently blocked with 3% BSA for 2?h at 4?C. Subsequently, the wells were washed five occasions with PBS and 100?L of F8 and G10 phage clones (109?PFU) was added for 1?h at RT with gentle agitation. Later on the plate was washed five occasions with PBS and the HRPCanti\M13\mAB (1?:?5000?v/v) was added and incubated at RT for 1?h. Next, the plate was washed 10 occasions with PBS and substrate TMB was utilized for detection with the absorbance measured at 450?nm. Measurements were performed three times, each time in triplicate. Competitive ELISA An ELISA plate was coated with FGFR1\Fc as for quantitative ELISA. The wells were washed five occasions with PBS and 100?L of FGF1 (10?gmL?1) and phage clone (109?PFU) mix or phage alone was added for 1?h at 4?C with gentle agitation. After incubation the plate was washed five occasions with PBS and the detection with HRPCanti\M13\mAB (1?:?5000?v/v) and TMB substrate was performed while before. Measurements were performed three times, each time in triplicate. Peptide synthesis and disulfide bridge formation Peptides were synthesized by hand with C\terminal amidation on Fmoc\solid phase according to standard strategy. The purity of acquired products was verified using.The peptide identified by phage display screening and after reformatting into Fc\fusion protein (peptibody), was approved in 2008 from the FDA for treatment of?chronic idiopathic thrombocytopenic purpura. Like a molecular target, we have chosen FGFR1, since it can regulate tumor growth and stimulate angiogenesis, either by aberrant signaling or by overexpression within the malignancy cell surface, as reported for multiple lung and breast cancers. for further development for potential use in treatments against FGFR1\expressing malignancy cells. BL21(DE3)pLysS strain at 37?C and purified on a heparinCSepharose CL\6B resin according to described protocols 23, 24. Phage display biopanning A 96\well plate was coated with 100?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6) and then blocked with BSA for 2?h at 4?C. Later on the wells were washed five occasions with TBST (0.1% Tween\20 in Tris\buffered saline). The original library was diluted in TBST (final concentration 2??1011 plaque forming models, PFU) and 100?L was added to each coated well for 2?h at 4?C with gentle agitation. After washing the plate 10 occasions with TBST, the bounded phages were eluted with 0.2?m glycine/HCl (pH 2.2) and neutralized with 1?m Tris/HCl (pH 9.1). The eluted phages were then amplified, precipitated with polyethylene glycol/NaCl and titrated relating to standard protocol (NEB). In the second round of selection there was an additional counterselection step for the Fc fragment. Fc at a concentration of 50?gmL?1 was immobilized in wells and blocked with 5% non\fat milk. Subsequently, the phages (2??1011?PFU) were incubated with Fc for 1?h at 4?C. Later on, phage clones were transferred to wells with FGFR1\Fc (70?gmL?1) and incubated for 1?h at 4?C with gentle agitation. After each round of selection, amplified phages were diluted and utilized for the next round of biopanning. In the third round of selection, the concentration of immobilized FGFR1\Fc and Fc was decreased down to 50 and 20?gmL?1, respectively. Additionally after the last round of selection, binding phages were eluted with 100 molar excess of FGF1 over applied phage library (2??1011?PFU). ELISA screening ELISA was conducted after the third round of selection. The 96\well Maxisorp F plate was coated with FGFR1\Fc (5?g per well), incubated at 4?C overnight and additionally blocked with 3% BSA for 2?h at 4?C. After washing with TBST (0.2% Tween\20) inoculated phage clones were added to the wells and incubated for 2?h at 4?C. Afterward the plate was washed five occasions and horseradish peroxidase (HRP)Canti\M13\monoclonal antibody (mAB) (1?:?5000?v/v, no. 27\9421\01; GE Healthcare, Chicago, IL, USA) was added to each well and incubated at room heat (RT) for 1?h. Subsequently, the plate was washed four occasions and 3,3,5,5\tetramethylbenzidine (TMB) was used for detection with the absorbance measured at 450?nm. Similarly, for the estimation of the level of Fc\binding, the plate was coated with Fc (5?g per well) and treated with phage clones as above. Quantitative ELISA The 96\well plate was coated with 50?gmL?1 FGFR1\Fc overnight at 4?C in 0.1?m NaHCO3 (pH 8.6), washed three times with PBS and subsequently blocked with 3% BSA for 2?h at 4?C. Subsequently, the wells were washed five occasions with PBS and 100?L of F8 and G10 phage clones (109?PFU) was added for 1?h at RT with gentle agitation. Afterwards the plate was washed five occasions with PBS and the HRPCanti\M13\mAB (1?:?5000?v/v) was added and incubated at RT for 1?h. Bitopertin (R enantiomer) Next, the plate was washed 10 occasions with PBS and substrate TMB was used for detection with the absorbance measured at 450?nm. Measurements were performed three times, each time in triplicate. Competitive ELISA An ELISA plate was coated with FGFR1\Fc as for quantitative ELISA. The wells were washed five occasions with PBS and 100?L of FGF1 (10?gmL?1) and phage clone (109?PFU) mix or phage alone was added for 1?h at 4?C with gentle agitation. After incubation the plate was washed five occasions with PBS and the detection with HRPCanti\M13\mAB (1?:?5000?v/v) and TMB substrate was performed as before. Measurements were performed three times, each time in triplicate. Peptide synthesis and disulfide bridge formation Peptides were synthesized manually with C\terminal amidation on Fmoc\solid phase according to standard strategy. The purity of obtained products was verified using RP\HPLC and the proper molar masses of synthesized peptides were confirmed by mass spectrometry. Disulfide bridge formation was optimized and oxidation folding in redox buffer protocol proved to be the most efficient method. Peptides at a final concentration of 0.1?mgmL?1 were dissolved in Milli\Q water (Merck Millipore; Burlington, MA, USA) at pH 3 and mixed with 4?m urea, 300?m reduced glutathione and 150?m oxidized glutathione. The pH was then.
(B) Quantitative ELISA was performed with either immobilized FGFR1\Fc or Fc, to determine peptide specificity towards FGFR1 (C) Competitive ELISA detected decreased F8 clone binding to FGFR1 in the presence of FGF1, suggesting the possibility of overlapping binding interface
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