Structured Review

Millipore cacl2
Cross‐linking of fibrin by full fength rFXIIIA and fragments. Clots were formed with fibrinogen, thrombin, <t>CaCl2,</t> and either full length rFXIIIA (lane 2), fragment TB 1 (lacking barrel 2; lane 3), TCC (lacking barrels 1 and 2; lane 4), TBS (lacking catalytic core and barrels 1 and 2; lane 5), or a control without any form of FXIII (lane 6). After 30 min, samples were run on an SDS ‐ PAGE gel under reducing conditions (A). The mean density of the different bands was determined relative to the control for that peptide chain or cross‐linked structure (B). FXIIIA , full length FXIII A subunit; TB 1, full length FXIII A subunit lacking barrel 2; TBS , full length FXIII A subunit lacking barrels 1 and 2, and catalytic core; TCC , full length FXIII A subunit lacking barrels 1 and 2.
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Images

1) Product Images from "The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit"

Article Title: The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit

Journal: Journal of Thrombosis and Haemostasis

doi: 10.1111/jth.14128

Cross‐linking of fibrin by full fength rFXIIIA and fragments. Clots were formed with fibrinogen, thrombin, CaCl2, and either full length rFXIIIA (lane 2), fragment TB 1 (lacking barrel 2; lane 3), TCC (lacking barrels 1 and 2; lane 4), TBS (lacking catalytic core and barrels 1 and 2; lane 5), or a control without any form of FXIII (lane 6). After 30 min, samples were run on an SDS ‐ PAGE gel under reducing conditions (A). The mean density of the different bands was determined relative to the control for that peptide chain or cross‐linked structure (B). FXIIIA , full length FXIII A subunit; TB 1, full length FXIII A subunit lacking barrel 2; TBS , full length FXIII A subunit lacking barrels 1 and 2, and catalytic core; TCC , full length FXIII A subunit lacking barrels 1 and 2.
Figure Legend Snippet: Cross‐linking of fibrin by full fength rFXIIIA and fragments. Clots were formed with fibrinogen, thrombin, CaCl2, and either full length rFXIIIA (lane 2), fragment TB 1 (lacking barrel 2; lane 3), TCC (lacking barrels 1 and 2; lane 4), TBS (lacking catalytic core and barrels 1 and 2; lane 5), or a control without any form of FXIII (lane 6). After 30 min, samples were run on an SDS ‐ PAGE gel under reducing conditions (A). The mean density of the different bands was determined relative to the control for that peptide chain or cross‐linked structure (B). FXIIIA , full length FXIII A subunit; TB 1, full length FXIII A subunit lacking barrel 2; TBS , full length FXIII A subunit lacking barrels 1 and 2, and catalytic core; TCC , full length FXIII A subunit lacking barrels 1 and 2.

Techniques Used: SDS Page

Effect of recombinant Factor XIIIA and fragments on turbidity and clot lysis. Polymerisation of FXIIIA depleted fibrinogen was initiated by the addition of thrombin, CaCl2, and either full length rFXIIIA (black square), fragment TB 1 (lacking barrel 2; dark grey diamond), TCC (lacking barrels 1 and 2; mid‐grey triangle), TBS (lacking catalytic core and barrels 1 and 2; light grey circles), or a control (buffer; very light grey crosses). Generation of turbidity was measured every 12 s for 60 min and results shown are mean values of triplicate experiments (A). Only clots with full‐length FXIII ‐A had a significant decrease in final maximum absorbance compared to control ( n = 3, P
Figure Legend Snippet: Effect of recombinant Factor XIIIA and fragments on turbidity and clot lysis. Polymerisation of FXIIIA depleted fibrinogen was initiated by the addition of thrombin, CaCl2, and either full length rFXIIIA (black square), fragment TB 1 (lacking barrel 2; dark grey diamond), TCC (lacking barrels 1 and 2; mid‐grey triangle), TBS (lacking catalytic core and barrels 1 and 2; light grey circles), or a control (buffer; very light grey crosses). Generation of turbidity was measured every 12 s for 60 min and results shown are mean values of triplicate experiments (A). Only clots with full‐length FXIII ‐A had a significant decrease in final maximum absorbance compared to control ( n = 3, P

Techniques Used: Recombinant, Lysis

2) Product Images from "The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit"

Article Title: The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit

Journal: Journal of Thrombosis and Haemostasis

doi: 10.1111/jth.14128

Cross‐linking of fibrin by full fength rFXIIIA and fragments. Clots were formed with fibrinogen, thrombin, CaCl2, and either full length rFXIIIA (lane 2), fragment TB 1 (lacking barrel 2; lane 3), TCC (lacking barrels 1 and 2; lane 4), TBS (lacking catalytic core and barrels 1 and 2; lane 5), or a control without any form of FXIII (lane 6). After 30 min, samples were run on an SDS ‐ PAGE gel under reducing conditions (A). The mean density of the different bands was determined relative to the control for that peptide chain or cross‐linked structure (B). FXIIIA , full length FXIII A subunit; TB 1, full length FXIII A subunit lacking barrel 2; TBS , full length FXIII A subunit lacking barrels 1 and 2, and catalytic core; TCC , full length FXIII A subunit lacking barrels 1 and 2.
Figure Legend Snippet: Cross‐linking of fibrin by full fength rFXIIIA and fragments. Clots were formed with fibrinogen, thrombin, CaCl2, and either full length rFXIIIA (lane 2), fragment TB 1 (lacking barrel 2; lane 3), TCC (lacking barrels 1 and 2; lane 4), TBS (lacking catalytic core and barrels 1 and 2; lane 5), or a control without any form of FXIII (lane 6). After 30 min, samples were run on an SDS ‐ PAGE gel under reducing conditions (A). The mean density of the different bands was determined relative to the control for that peptide chain or cross‐linked structure (B). FXIIIA , full length FXIII A subunit; TB 1, full length FXIII A subunit lacking barrel 2; TBS , full length FXIII A subunit lacking barrels 1 and 2, and catalytic core; TCC , full length FXIII A subunit lacking barrels 1 and 2.

Techniques Used: SDS Page

Effect of recombinant Factor XIIIA and fragments on turbidity and clot lysis. Polymerisation of FXIIIA depleted fibrinogen was initiated by the addition of thrombin, CaCl2, and either full length rFXIIIA (black square), fragment TB 1 (lacking barrel 2; dark grey diamond), TCC (lacking barrels 1 and 2; mid‐grey triangle), TBS (lacking catalytic core and barrels 1 and 2; light grey circles), or a control (buffer; very light grey crosses). Generation of turbidity was measured every 12 s for 60 min and results shown are mean values of triplicate experiments (A). Only clots with full‐length FXIII ‐A had a significant decrease in final maximum absorbance compared to control ( n = 3, P
Figure Legend Snippet: Effect of recombinant Factor XIIIA and fragments on turbidity and clot lysis. Polymerisation of FXIIIA depleted fibrinogen was initiated by the addition of thrombin, CaCl2, and either full length rFXIIIA (black square), fragment TB 1 (lacking barrel 2; dark grey diamond), TCC (lacking barrels 1 and 2; mid‐grey triangle), TBS (lacking catalytic core and barrels 1 and 2; light grey circles), or a control (buffer; very light grey crosses). Generation of turbidity was measured every 12 s for 60 min and results shown are mean values of triplicate experiments (A). Only clots with full‐length FXIII ‐A had a significant decrease in final maximum absorbance compared to control ( n = 3, P

Techniques Used: Recombinant, Lysis

3) Product Images from "Adipose Stem Cell Microbeads as Production Sources for Chondrogenic Growth Factors"

Article Title: Adipose Stem Cell Microbeads as Production Sources for Chondrogenic Growth Factors

Journal: Journal of Stem Cells & Regenerative Medicine

doi:

Effect of Divalent Crosslinks in ASC Microbead on Growth Factor mRNA Levels and Production. (A) mRNA levels and (B) growth factor secretion/DNA content on day 7 from ASC microbeads crosslinked in CaCl2 Ca++) or BaCl2 (Ba++), cultured in growth medium and normalized to ASCs on tissue culture polystyrene (TCPS). (C) Growth factor retained within microbeads normalized to DNA content (n = 6 samples, mean±SE, *p
Figure Legend Snippet: Effect of Divalent Crosslinks in ASC Microbead on Growth Factor mRNA Levels and Production. (A) mRNA levels and (B) growth factor secretion/DNA content on day 7 from ASC microbeads crosslinked in CaCl2 Ca++) or BaCl2 (Ba++), cultured in growth medium and normalized to ASCs on tissue culture polystyrene (TCPS). (C) Growth factor retained within microbeads normalized to DNA content (n = 6 samples, mean±SE, *p

Techniques Used: Cell Culture

4) Product Images from "Adipose Stem Cell Microbeads as Production Sources for Chondrogenic Growth Factors"

Article Title: Adipose Stem Cell Microbeads as Production Sources for Chondrogenic Growth Factors

Journal: Journal of Stem Cells & Regenerative Medicine

doi:

Effect of Divalent Crosslinks in ASC Microbead on Growth Factor mRNA Levels and Production. (A) mRNA levels and (B) growth factor secretion/DNA content on day 7 from ASC microbeads crosslinked in CaCl2 Ca++) or BaCl2 (Ba++), cultured in growth medium and normalized to ASCs on tissue culture polystyrene (TCPS). (C) Growth factor retained within microbeads normalized to DNA content (n = 6 samples, mean±SE, *p
Figure Legend Snippet: Effect of Divalent Crosslinks in ASC Microbead on Growth Factor mRNA Levels and Production. (A) mRNA levels and (B) growth factor secretion/DNA content on day 7 from ASC microbeads crosslinked in CaCl2 Ca++) or BaCl2 (Ba++), cultured in growth medium and normalized to ASCs on tissue culture polystyrene (TCPS). (C) Growth factor retained within microbeads normalized to DNA content (n = 6 samples, mean±SE, *p

Techniques Used: Cell Culture

Related Articles

Clone Assay:

Article Title: Atomic structure of the eukaryotic intramembrane Ras methyltransferase ICMT
Article Snippet: Paragraph title: Cloning, expression, and purification of ICMT ... Approximately 0.4 ml of YL1/2 antibody beads were added to the sample for each 1 g of P. pastoris cells and the mixture was rotated at room temperature for 1 h. Beads were collected on a column, washed with 4 column volumes of buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and the protein was eluted with buffer containing 100 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and 5 mM Asp-Phe peptide (Sigma-Aldrich) or Glu-Glu-Phe peptide (Peptide 2.0).

Zymography:

Article Title: Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri
Article Snippet: Paragraph title: 4.4.4. Gelatin Zymography ... Subsequent to the electrophoresis, the gel was washed twice for 30 min at room temperature in 2.5% Triton X-100 (Sigma-Aldrich) to remove the SDS and incubated at 37 °C for 18 h in one of the following buffers: 0.05 M sodium citrate pH 4.0, pH 5.0 and pH 6.0; 0.05 M Tris-HCl pH 7.0, pH 8.0, pH 9.0 and pH 10.0; and in the presence of ions and other chemicals as 50 mM Tris-HCl pH 8.0; 50 mM Tris-HCl and 10 mM CaCl2 (Sigma-Aldrich) pH 8.0; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2 , 0.002% CHAPS (Sigma-Aldrich) and 10 mM EDTA pH 8.0; and 50 mM Tris-HCl, 1 mM CaCl2 and 1 mM ZnSO4 (Sigma-Aldrich) pH 8.0.

Blocking Assay:

Article Title: The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit
Article Snippet: .. Briefly, microtiter plates were coated with 40 μg mL−1 human fibrinogen (Enzyme Research Laboratories) at 37 °C for 1 h. After blocking with 1% BSA, plates were incubated with 1 U mL−1 human thrombin (Calbiochem) and 5 mm CaCl2 to convert fibrinogen to fibrin, and then treated in triplicate with 3.5 nm recombinant FXIII‐A sample, 10 μg mL−1 α2 ‐antiplasmin (Calbiochem), 1 U mL−1 human thrombin, 0.1 mm DTT, and 1 mm CaCl2 . .. Incorporation of α2 ‐antiplasmin was stopped with 133 mm EDTA over a time course of 50 min. Crosslinking of the α2 ‐antiplasmin into the fibrin by recombinant FXIII was detected by the use of goat anti‐human α2 ‐antiplasmin antibody with a horseradish peroxidase conjugate (Enzyme Research Laboratories) and o ‐phenylenediamine (OPD) (Dako, Ely, UK).

Article Title: Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
Article Snippet: .. The blots were washed three times with blocking buffer with 2 mM CaCl2 for 10 min each and then incubated with blocking buffer with 2 mM CaCl2 and a polyclonal anti-rabbit secondary antibody conjugated to HRP (1:15,000) (Sigma, A9169) for 2 h at RT. .. The blot was washed three times with blocking buffer with 2 mM CaCl2 for 5 min each at RT.

Electrophoresis:

Article Title: Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri
Article Snippet: .. Subsequent to the electrophoresis, the gel was washed twice for 30 min at room temperature in 2.5% Triton X-100 (Sigma-Aldrich) to remove the SDS and incubated at 37 °C for 18 h in one of the following buffers: 0.05 M sodium citrate pH 4.0, pH 5.0 and pH 6.0; 0.05 M Tris-HCl pH 7.0, pH 8.0, pH 9.0 and pH 10.0; and in the presence of ions and other chemicals as 50 mM Tris-HCl pH 8.0; 50 mM Tris-HCl and 10 mM CaCl2 (Sigma-Aldrich) pH 8.0; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2 , 0.002% CHAPS (Sigma-Aldrich) and 10 mM EDTA pH 8.0; and 50 mM Tris-HCl, 1 mM CaCl2 and 1 mM ZnSO4 (Sigma-Aldrich) pH 8.0. .. The gels were stained with R-250 Coomassie blue and gelatin proteolysis activity detected as colorless bands in the otherwise blue gel.

Incubation:

Article Title: The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit
Article Snippet: .. Briefly, microtiter plates were coated with 40 μg mL−1 human fibrinogen (Enzyme Research Laboratories) at 37 °C for 1 h. After blocking with 1% BSA, plates were incubated with 1 U mL−1 human thrombin (Calbiochem) and 5 mm CaCl2 to convert fibrinogen to fibrin, and then treated in triplicate with 3.5 nm recombinant FXIII‐A sample, 10 μg mL−1 α2 ‐antiplasmin (Calbiochem), 1 U mL−1 human thrombin, 0.1 mm DTT, and 1 mm CaCl2 . .. Incorporation of α2 ‐antiplasmin was stopped with 133 mm EDTA over a time course of 50 min. Crosslinking of the α2 ‐antiplasmin into the fibrin by recombinant FXIII was detected by the use of goat anti‐human α2 ‐antiplasmin antibody with a horseradish peroxidase conjugate (Enzyme Research Laboratories) and o ‐phenylenediamine (OPD) (Dako, Ely, UK).

Article Title: Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
Article Snippet: .. The blots were washed three times with blocking buffer with 2 mM CaCl2 for 10 min each and then incubated with blocking buffer with 2 mM CaCl2 and a polyclonal anti-rabbit secondary antibody conjugated to HRP (1:15,000) (Sigma, A9169) for 2 h at RT. .. The blot was washed three times with blocking buffer with 2 mM CaCl2 for 5 min each at RT.

Article Title: Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri
Article Snippet: .. Subsequent to the electrophoresis, the gel was washed twice for 30 min at room temperature in 2.5% Triton X-100 (Sigma-Aldrich) to remove the SDS and incubated at 37 °C for 18 h in one of the following buffers: 0.05 M sodium citrate pH 4.0, pH 5.0 and pH 6.0; 0.05 M Tris-HCl pH 7.0, pH 8.0, pH 9.0 and pH 10.0; and in the presence of ions and other chemicals as 50 mM Tris-HCl pH 8.0; 50 mM Tris-HCl and 10 mM CaCl2 (Sigma-Aldrich) pH 8.0; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2 , 0.002% CHAPS (Sigma-Aldrich) and 10 mM EDTA pH 8.0; and 50 mM Tris-HCl, 1 mM CaCl2 and 1 mM ZnSO4 (Sigma-Aldrich) pH 8.0. .. The gels were stained with R-250 Coomassie blue and gelatin proteolysis activity detected as colorless bands in the otherwise blue gel.

Article Title: Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME
Article Snippet: 50% of the TEV eluate was incubated with Calmodulin Sepharose™ 4B (GE Healthcare), whereas the other 50% of the TEV eluate was incubated with Anti-FLAG® M2 Affinity Gel (Flag-beads, Sigma) at 4°C for 60 min. After washing the Calmodulin beads with 2 ml buffer containing 2 mM CaCl2 , followed by a second washing step with 5 ml 2 mM CaCl2 alone, proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Nap1-TAP). .. After washing the Calmodulin beads with 2 ml buffer containing 2 mM CaCl2 , followed by a second washing step with 5 ml 2 mM CaCl2 alone, proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a depleted).

Stripping Membranes:

Article Title: Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
Article Snippet: The beads were washed three times with 50 mM HEPES (pH 7.4) using the magnetic strip and then resuspended in 90 μl of 0.1 M glycine (pH 2.5). .. The blots were washed three times with blocking buffer with 2 mM CaCl2 for 10 min each and then incubated with blocking buffer with 2 mM CaCl2 and a polyclonal anti-rabbit secondary antibody conjugated to HRP (1:15,000) (Sigma, A9169) for 2 h at RT.

Activity Assay:

Article Title: The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit
Article Snippet: Paragraph title: Determination of protein activity by α2 ‐antiplasmin incorporation ... Briefly, microtiter plates were coated with 40 μg mL−1 human fibrinogen (Enzyme Research Laboratories) at 37 °C for 1 h. After blocking with 1% BSA, plates were incubated with 1 U mL−1 human thrombin (Calbiochem) and 5 mm CaCl2 to convert fibrinogen to fibrin, and then treated in triplicate with 3.5 nm recombinant FXIII‐A sample, 10 μg mL−1 α2 ‐antiplasmin (Calbiochem), 1 U mL−1 human thrombin, 0.1 mm DTT, and 1 mm CaCl2 .

Article Title: Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri
Article Snippet: Subsequent to the electrophoresis, the gel was washed twice for 30 min at room temperature in 2.5% Triton X-100 (Sigma-Aldrich) to remove the SDS and incubated at 37 °C for 18 h in one of the following buffers: 0.05 M sodium citrate pH 4.0, pH 5.0 and pH 6.0; 0.05 M Tris-HCl pH 7.0, pH 8.0, pH 9.0 and pH 10.0; and in the presence of ions and other chemicals as 50 mM Tris-HCl pH 8.0; 50 mM Tris-HCl and 10 mM CaCl2 (Sigma-Aldrich) pH 8.0; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2 , 0.002% CHAPS (Sigma-Aldrich) and 10 mM EDTA pH 8.0; and 50 mM Tris-HCl, 1 mM CaCl2 and 1 mM ZnSO4 (Sigma-Aldrich) pH 8.0. .. The gels were stained with R-250 Coomassie blue and gelatin proteolysis activity detected as colorless bands in the otherwise blue gel.

Expressing:

Article Title: Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME
Article Snippet: Nap1-TAP Rps6a-Flag split purification Yeast cells expressing Nap1-TAP Rps6a-Flag were grown at 30°C in 8 l YPD to an OD600 of 2. .. After washing the Calmodulin beads with 2 ml buffer containing 2 mM CaCl2 , followed by a second washing step with 5 ml 2 mM CaCl2 alone, proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a depleted).

Article Title: Use of transfected Drosophila S2 cells to study NK cell activation
Article Snippet: CaCl2 for calcium phosphate transfection: 2 M CaCl2 (Sigma Aldrich) in water, sterilized by filtration through a 0.45 µm filter. .. 6-well tissue culture plates (Costar, Corning). pAc5.1-V5-His: plasmid for constitutive expression in S2 cells using the Drosophila Actin 5 promoter (Requires a licensing agreement with Invitrogen). pRmHa3: plasmid for inducible expression in S2 cells using the metallothionein promoter (available to not-for-profit labs from the Drosophila Genome Research Center, Bloomington IN).

Article Title: Atomic structure of the eukaryotic intramembrane Ras methyltransferase ICMT
Article Snippet: Paragraph title: Cloning, expression, and purification of ICMT ... Approximately 0.4 ml of YL1/2 antibody beads were added to the sample for each 1 g of P. pastoris cells and the mixture was rotated at room temperature for 1 h. Beads were collected on a column, washed with 4 column volumes of buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and the protein was eluted with buffer containing 100 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and 5 mM Asp-Phe peptide (Sigma-Aldrich) or Glu-Glu-Phe peptide (Peptide 2.0).

Western Blot:

Article Title: Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
Article Snippet: The blots were washed three times with blocking buffer with 2 mM CaCl2 for 10 min each and then incubated with blocking buffer with 2 mM CaCl2 and a polyclonal anti-rabbit secondary antibody conjugated to HRP (1:15,000) (Sigma, A9169) for 2 h at RT. .. The blots were incubated with GE Healthcare ECL Western blotting detection kit and imaged with a BIO-RAD ChemiDoc Touch imaging system for chemiluminescence.

Article Title: Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME
Article Snippet: After washing the Calmodulin beads with 2 ml buffer containing 2 mM CaCl2 , followed by a second washing step with 5 ml 2 mM CaCl2 alone, proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a depleted). .. Protein samples were dried by SpeedVac® , dissolved in SDS sample buffer, and separated on NuPAGE™ 4–12% Bis–Tris gels (Invitrogen) followed by NOVEX® Colloidal Blue Staining Kit (Invitrogen) and western blotting.

Filtration:

Article Title: Use of transfected Drosophila S2 cells to study NK cell activation
Article Snippet: .. CaCl2 for calcium phosphate transfection: 2 M CaCl2 (Sigma Aldrich) in water, sterilized by filtration through a 0.45 µm filter. .. 6-well tissue culture plates (Costar, Corning). pAc5.1-V5-His: plasmid for constitutive expression in S2 cells using the Drosophila Actin 5 promoter (Requires a licensing agreement with Invitrogen). pRmHa3: plasmid for inducible expression in S2 cells using the metallothionein promoter (available to not-for-profit labs from the Drosophila Genome Research Center, Bloomington IN).

Transformation Assay:

Article Title: Atomic structure of the eukaryotic intramembrane Ras methyltransferase ICMT
Article Snippet: Transformation into P. pastoris , expression, and cryo-lysis were performed as previously described . .. Approximately 0.4 ml of YL1/2 antibody beads were added to the sample for each 1 g of P. pastoris cells and the mixture was rotated at room temperature for 1 h. Beads were collected on a column, washed with 4 column volumes of buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and the protein was eluted with buffer containing 100 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and 5 mM Asp-Phe peptide (Sigma-Aldrich) or Glu-Glu-Phe peptide (Peptide 2.0).

Transfection:

Article Title: Use of transfected Drosophila S2 cells to study NK cell activation
Article Snippet: .. CaCl2 for calcium phosphate transfection: 2 M CaCl2 (Sigma Aldrich) in water, sterilized by filtration through a 0.45 µm filter. .. 6-well tissue culture plates (Costar, Corning). pAc5.1-V5-His: plasmid for constitutive expression in S2 cells using the Drosophila Actin 5 promoter (Requires a licensing agreement with Invitrogen). pRmHa3: plasmid for inducible expression in S2 cells using the metallothionein promoter (available to not-for-profit labs from the Drosophila Genome Research Center, Bloomington IN).

Immunoprecipitation:

Article Title: Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
Article Snippet: Paragraph title: Immunoprecipitation and lipid dot blot assay ... The blots were washed three times with blocking buffer with 2 mM CaCl2 for 10 min each and then incubated with blocking buffer with 2 mM CaCl2 and a polyclonal anti-rabbit secondary antibody conjugated to HRP (1:15,000) (Sigma, A9169) for 2 h at RT.

Protease Inhibitor:

Article Title: Atomic structure of the eukaryotic intramembrane Ras methyltransferase ICMT
Article Snippet: Lysed cells (40 g) were re-suspended in 200 ml of buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM tri(2-carboxyethyl)phosphine (TCEP) (Soltec Ventures; a 275 mM stock solution of TCEP was prepared in 1 M KOH to yield pH ~7.5), 2 mM CaCl2 , 25 µM AdoHcy, 0.15 mg/mL deoxyribonuclease I (DNase I) (Sigma-Aldrich), 1:1000 dilution of Protease Inhibitor Cocktail Set III (EDTA free, CalBiochem), 1 mM benzamidine (Sigma-Aldrich), 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) (Gold Biotechnology) and 1:1000 dilution of aprotinin (Sigma-Aldrich). .. Approximately 0.4 ml of YL1/2 antibody beads were added to the sample for each 1 g of P. pastoris cells and the mixture was rotated at room temperature for 1 h. Beads were collected on a column, washed with 4 column volumes of buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and the protein was eluted with buffer containing 100 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and 5 mM Asp-Phe peptide (Sigma-Aldrich) or Glu-Glu-Phe peptide (Peptide 2.0).

Transferring:

Article Title: Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons
Article Snippet: The external solution (ECS) contained (in mM): 145 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 (all Sigma), 10 glucose and 10 HEPES (Merck, Darmstadt, Germany), at pH 7.3 adjusted with NaOH (Merck), and electrodes were filled with internal solution (ICS, in mM): 140 KCl, 2 MgCl2 , 2 Na-ATP, 0.2 Na-GTP, 0.1 CaCl2 , 1 EGTA (all Sigma) and 10 HEPES (Merck), at pH 7.3 adjusted with KOH (Merck). .. The pipette solution was composed (in mM) of 45 KCl, 98 K-gluconate, 0.5 CaCl2 , 5 EGTA, 10 HEPES, 2 MgATP, 0.2 NaGTP, pH 7.3 adjusted with KOH (Merck).

Inhibition:

Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
Article Snippet: Paragraph title: Calcium carbonate precipitation rate inhibition assay ... Different concentrations of CaCl2 and NaHCO3 were prepared and filtered by a 0.22 μm MILLEXGP filter unit. rPfY2 was mixed with CaCl2 (Sigma-Aldrich; 100 μl, 100 mM) in a 96-well COSMO plate.

Imaging:

Article Title: Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
Article Snippet: The blots were washed three times with blocking buffer with 2 mM CaCl2 for 10 min each and then incubated with blocking buffer with 2 mM CaCl2 and a polyclonal anti-rabbit secondary antibody conjugated to HRP (1:15,000) (Sigma, A9169) for 2 h at RT. .. The blots were incubated with GE Healthcare ECL Western blotting detection kit and imaged with a BIO-RAD ChemiDoc Touch imaging system for chemiluminescence.

Binding Assay:

Article Title: Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME
Article Snippet: After binding of the TEV eluate to Anti-FLAG® M2 Affinity Gel the supernatant was collected and bound to Calmodulin Sepharose™ 4B at 4°C for 60 min after adding 2 mM CaCl2 , whereas the Anti-FLAG® M2 Affinity Gel was washed with lysis buffer, and proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a-enriched). .. After washing the Calmodulin beads with 2 ml buffer containing 2 mM CaCl2 , followed by a second washing step with 5 ml 2 mM CaCl2 alone, proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a depleted).

Isolation:

Article Title: Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons
Article Snippet: Ionic currents were recorded from isolated DRG neurons in the whole-cell voltage-clamp configuration at −60 mV holding potential, unless otherwise indicated. .. The external solution (ECS) contained (in mM): 145 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 (all Sigma), 10 glucose and 10 HEPES (Merck, Darmstadt, Germany), at pH 7.3 adjusted with NaOH (Merck), and electrodes were filled with internal solution (ICS, in mM): 140 KCl, 2 MgCl2 , 2 Na-ATP, 0.2 Na-GTP, 0.1 CaCl2 , 1 EGTA (all Sigma) and 10 HEPES (Merck), at pH 7.3 adjusted with KOH (Merck).

Article Title: Adipose HuR protects against diet-induced obesity and insulin resistance
Article Snippet: .. Stromal vascular fraction isolation Mice at age 6−8 weeks were sacrificed and epididymal adipose tissues were removed, minced in phosphate-buffered saline (PBS) containing 10 mM CaCl2 , and digested at 37 °C for 60 min in DMEM containing 10 mM CaCl2 , 1.5 units ml−1 collagenase D (Sigma, 11088866001), and 2.4 units ml−1 dispase II (Sigma, D4693). ..

Purification:

Article Title: Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
Article Snippet: The blots were washed three times with blocking buffer with 2 mM CaCl2 for 10 min each and then incubated with blocking buffer with 2 mM CaCl2 and a polyclonal anti-rabbit secondary antibody conjugated to HRP (1:15,000) (Sigma, A9169) for 2 h at RT. .. Purified GST-CpnA (0.5 μg/mL), instead of the eluate, was also used as a control in the lipid dot blot assay.

Article Title: Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME
Article Snippet: Paragraph title: Nap1-TAP Rps6a-Flag split purification ... After washing the Calmodulin beads with 2 ml buffer containing 2 mM CaCl2 , followed by a second washing step with 5 ml 2 mM CaCl2 alone, proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a depleted).

Article Title: Atomic structure of the eukaryotic intramembrane Ras methyltransferase ICMT
Article Snippet: Paragraph title: Cloning, expression, and purification of ICMT ... Approximately 0.4 ml of YL1/2 antibody beads were added to the sample for each 1 g of P. pastoris cells and the mixture was rotated at room temperature for 1 h. Beads were collected on a column, washed with 4 column volumes of buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and the protein was eluted with buffer containing 100 mM Tris-HCl, pH 7.5, 150 mM KCl, 2 mM TCEP, 2 mM CaCl2 , 25 µM AdoHcy, 1 mM DMNG, and 5 mM Asp-Phe peptide (Sigma-Aldrich) or Glu-Glu-Phe peptide (Peptide 2.0).

Dot Blot:

Article Title: Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane
Article Snippet: Paragraph title: Immunoprecipitation and lipid dot blot assay ... The blots were washed three times with blocking buffer with 2 mM CaCl2 for 10 min each and then incubated with blocking buffer with 2 mM CaCl2 and a polyclonal anti-rabbit secondary antibody conjugated to HRP (1:15,000) (Sigma, A9169) for 2 h at RT.

Recombinant:

Article Title: The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit. The role of β‐barrels 1 and 2 in the enzymatic activity of factor XIII A‐subunit
Article Snippet: .. Briefly, microtiter plates were coated with 40 μg mL−1 human fibrinogen (Enzyme Research Laboratories) at 37 °C for 1 h. After blocking with 1% BSA, plates were incubated with 1 U mL−1 human thrombin (Calbiochem) and 5 mm CaCl2 to convert fibrinogen to fibrin, and then treated in triplicate with 3.5 nm recombinant FXIII‐A sample, 10 μg mL−1 α2 ‐antiplasmin (Calbiochem), 1 U mL−1 human thrombin, 0.1 mm DTT, and 1 mm CaCl2 . .. Incorporation of α2 ‐antiplasmin was stopped with 133 mm EDTA over a time course of 50 min. Crosslinking of the α2 ‐antiplasmin into the fibrin by recombinant FXIII was detected by the use of goat anti‐human α2 ‐antiplasmin antibody with a horseradish peroxidase conjugate (Enzyme Research Laboratories) and o ‐phenylenediamine (OPD) (Dako, Ely, UK).

Staining:

Article Title: Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri
Article Snippet: Subsequent to the electrophoresis, the gel was washed twice for 30 min at room temperature in 2.5% Triton X-100 (Sigma-Aldrich) to remove the SDS and incubated at 37 °C for 18 h in one of the following buffers: 0.05 M sodium citrate pH 4.0, pH 5.0 and pH 6.0; 0.05 M Tris-HCl pH 7.0, pH 8.0, pH 9.0 and pH 10.0; and in the presence of ions and other chemicals as 50 mM Tris-HCl pH 8.0; 50 mM Tris-HCl and 10 mM CaCl2 (Sigma-Aldrich) pH 8.0; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2 , 0.002% CHAPS (Sigma-Aldrich) and 10 mM EDTA pH 8.0; and 50 mM Tris-HCl, 1 mM CaCl2 and 1 mM ZnSO4 (Sigma-Aldrich) pH 8.0. .. The gels were stained with R-250 Coomassie blue and gelatin proteolysis activity detected as colorless bands in the otherwise blue gel.

Article Title: Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME
Article Snippet: After washing the Calmodulin beads with 2 ml buffer containing 2 mM CaCl2 , followed by a second washing step with 5 ml 2 mM CaCl2 alone, proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a depleted). .. Protein samples were dried by SpeedVac® , dissolved in SDS sample buffer, and separated on NuPAGE™ 4–12% Bis–Tris gels (Invitrogen) followed by NOVEX® Colloidal Blue Staining Kit (Invitrogen) and western blotting.

Mouse Assay:

Article Title: Adipose HuR protects against diet-induced obesity and insulin resistance
Article Snippet: .. Stromal vascular fraction isolation Mice at age 6−8 weeks were sacrificed and epididymal adipose tissues were removed, minced in phosphate-buffered saline (PBS) containing 10 mM CaCl2 , and digested at 37 °C for 60 min in DMEM containing 10 mM CaCl2 , 1.5 units ml−1 collagenase D (Sigma, 11088866001), and 2.4 units ml−1 dispase II (Sigma, D4693). ..

SDS Page:

Article Title: Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri
Article Snippet: Crude venom samples (5 µg) were separated by 12% SDS-PAGE containing 1% of the gelatin substrate (Sigma-Aldrich). .. Subsequent to the electrophoresis, the gel was washed twice for 30 min at room temperature in 2.5% Triton X-100 (Sigma-Aldrich) to remove the SDS and incubated at 37 °C for 18 h in one of the following buffers: 0.05 M sodium citrate pH 4.0, pH 5.0 and pH 6.0; 0.05 M Tris-HCl pH 7.0, pH 8.0, pH 9.0 and pH 10.0; and in the presence of ions and other chemicals as 50 mM Tris-HCl pH 8.0; 50 mM Tris-HCl and 10 mM CaCl2 (Sigma-Aldrich) pH 8.0; 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2 , 0.002% CHAPS (Sigma-Aldrich) and 10 mM EDTA pH 8.0; and 50 mM Tris-HCl, 1 mM CaCl2 and 1 mM ZnSO4 (Sigma-Aldrich) pH 8.0.

Plasmid Preparation:

Article Title: Use of transfected Drosophila S2 cells to study NK cell activation
Article Snippet: CaCl2 for calcium phosphate transfection: 2 M CaCl2 (Sigma Aldrich) in water, sterilized by filtration through a 0.45 µm filter. .. 6-well tissue culture plates (Costar, Corning). pAc5.1-V5-His: plasmid for constitutive expression in S2 cells using the Drosophila Actin 5 promoter (Requires a licensing agreement with Invitrogen). pRmHa3: plasmid for inducible expression in S2 cells using the metallothionein promoter (available to not-for-profit labs from the Drosophila Genome Research Center, Bloomington IN).

Selection:

Article Title: Use of transfected Drosophila S2 cells to study NK cell activation
Article Snippet: CaCl2 for calcium phosphate transfection: 2 M CaCl2 (Sigma Aldrich) in water, sterilized by filtration through a 0.45 µm filter. .. Plasmids for selection of transfectants, which include pNeoFly , pCoBlast (Invitrogen) and pCoHygro (Invitrogen).

Patch Clamp:

Article Title: Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl− Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons
Article Snippet: Electrophysiology Whole-cell patch-clamp recordings were performed using patch pipettes with a tip resistance of 2–4 MΩ as previously described (Camprubí-Robles et al., ). .. The external solution (ECS) contained (in mM): 145 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 (all Sigma), 10 glucose and 10 HEPES (Merck, Darmstadt, Germany), at pH 7.3 adjusted with NaOH (Merck), and electrodes were filled with internal solution (ICS, in mM): 140 KCl, 2 MgCl2 , 2 Na-ATP, 0.2 Na-GTP, 0.1 CaCl2 , 1 EGTA (all Sigma) and 10 HEPES (Merck), at pH 7.3 adjusted with KOH (Merck).

Spectrophotometry:

Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
Article Snippet: Calcium carbonate precipitation rate inhibition assay The inhibitory effects of rPfY2 on calcium carbonate precipitation rate was assessed by a spectrophotometer (Bio-rad 680, USA) in reference to Suzuki’s methods with minor modifications . .. Different concentrations of CaCl2 and NaHCO3 were prepared and filtered by a 0.22 μm MILLEXGP filter unit. rPfY2 was mixed with CaCl2 (Sigma-Aldrich; 100 μl, 100 mM) in a 96-well COSMO plate.

Concentration Assay:

Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
Article Snippet: Different concentrations of CaCl2 and NaHCO3 were prepared and filtered by a 0.22 μm MILLEXGP filter unit. rPfY2 was mixed with CaCl2 (Sigma-Aldrich; 100 μl, 100 mM) in a 96-well COSMO plate. .. The final concentration of rPfY2 was 2 μg/ml, 5 μg/ml and 10 μg/ml in experimental groups. rMBP and BSA were 10 μg/ml each.

Lysis:

Article Title: Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME
Article Snippet: After binding of the TEV eluate to Anti-FLAG® M2 Affinity Gel the supernatant was collected and bound to Calmodulin Sepharose™ 4B at 4°C for 60 min after adding 2 mM CaCl2 , whereas the Anti-FLAG® M2 Affinity Gel was washed with lysis buffer, and proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a-enriched). .. After washing the Calmodulin beads with 2 ml buffer containing 2 mM CaCl2 , followed by a second washing step with 5 ml 2 mM CaCl2 alone, proteins were eluted with 600 μl 0.8% ammonium hydroxide solution (Sigma) under rotation at room temperature for 20 min (Rps6a depleted).

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    Millipore tyrode s solution
    Tyrode S Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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