trna sepharose column  (Millipore)


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    Structured Review

    Millipore trna sepharose column
    In vitro production of Arabidopsis <t>tRNA</t> ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA <t>agarose</t> as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).
    Trna Sepharose Column, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trna sepharose column/product/Millipore
    Average 85 stars, based on 1 article reviews
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    1) Product Images from "Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins"

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki174

    In vitro production of Arabidopsis tRNA ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA agarose as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).
    Figure Legend Snippet: In vitro production of Arabidopsis tRNA ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA agarose as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).

    Techniques Used: In Vitro, Purification, Recombinant, Over Expression, Incubation, Plasmid Preparation, Chromatography, Flow Cytometry, Binding Assay, SDS-Gel, Staining

    Isolation of wheat germ tRNA ligase. ( A ) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. ( B ) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. ( C ) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc) 2 , 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10 4 c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.
    Figure Legend Snippet: Isolation of wheat germ tRNA ligase. ( A ) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. ( B ) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. ( C ) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc) 2 , 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10 4 c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.

    Techniques Used: Isolation, Purification, Activity Assay, Modification, Fractionation, Chromatography, Ligation, Incubation

    Gel filtration on Superdex™ 200 and adenylyltransferase activity of wheat germ tRNA ligase. ( A ) Partially purified tRNA ligase from the Source S15 column was subjected to gel filtration on Superdex™ 200. The column (HiLoad™ 16/60) was run with a flow rate of 1 ml/min. Fractions of 2 ml were collected. Aliquots of the elution fraction were analysed on a 7.5% polyacrylamide/0.1% SDS gel. The proteins were visualized by silver staining. ( B ) Appropriate aliquots of the indicated fractions were incubated in the presence of [α- 32 P]ATP for 15 min at 37°C. The ligase–[ 32 P]AMP adduct was detected by autoradiography of the dried gel. ( C ) The peak fractions from the tRNA Sepharose column were concentrated by ultrafiltration and 1/20 of this material was applied onto a 10% polyacrylamide/0.1% SDS gel and stained with Coomassie blue for analytical valuation. The arrows point to the position of the RNA ligase protein with an approximate molecular weight of 125 kDa. Protein size standards in kDa are indicated on the right.
    Figure Legend Snippet: Gel filtration on Superdex™ 200 and adenylyltransferase activity of wheat germ tRNA ligase. ( A ) Partially purified tRNA ligase from the Source S15 column was subjected to gel filtration on Superdex™ 200. The column (HiLoad™ 16/60) was run with a flow rate of 1 ml/min. Fractions of 2 ml were collected. Aliquots of the elution fraction were analysed on a 7.5% polyacrylamide/0.1% SDS gel. The proteins were visualized by silver staining. ( B ) Appropriate aliquots of the indicated fractions were incubated in the presence of [α- 32 P]ATP for 15 min at 37°C. The ligase–[ 32 P]AMP adduct was detected by autoradiography of the dried gel. ( C ) The peak fractions from the tRNA Sepharose column were concentrated by ultrafiltration and 1/20 of this material was applied onto a 10% polyacrylamide/0.1% SDS gel and stained with Coomassie blue for analytical valuation. The arrows point to the position of the RNA ligase protein with an approximate molecular weight of 125 kDa. Protein size standards in kDa are indicated on the right.

    Techniques Used: Filtration, Activity Assay, Purification, Flow Cytometry, SDS-Gel, Silver Staining, Incubation, Autoradiography, Staining, Molecular Weight

    Related Articles

    SDS-Gel:

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins
    Article Snippet: .. The pooled fractions from the tRNA Sepharose column were concentrated by ultrafiltration with Microcon 100 (Millipore, Schwalbach, Germany) and applied to a 10% polyacrylamide/0.1% SDS gel ( ). .. After a brief staining with Coomassie brilliant blue, ∼5 μg of the 125 kDa protein was excised from the gel.

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    Millipore polyacrylamide gel electrophoresis analysis
    siRNAs can be conjugated to a DT. ( A ) a DT was engineered to contain a free cysteine as a functional handle ( a DT-SH), protected by a SUMO tag and purified using a histidine (His) tag. ( B ) a DT was reacted with a PEG cross-linker containing both maleimide and DBCO functional groups to obtain DBCO-modified a DT ( a DT-DBCO). ( C ) The presence of the DBCO modification on a DT was confirmed by measuring the absorbance at 280 nm ( a DT) and 309 nm (DBCO). Curves shown are a DT before modification (blue) and after DBCO modification (red). A 280 , absorbance at 280 nm. ( D ) Azide-modified siRNA was reacted with the DBCO-functionalized a DT to obtain the a DT-siRNA conjugate. RT, room temperature. ( E ) Modification of the a DT with the siRNA was confirmed via <t>polyacrylamide</t> <t>gel</t> <t>electrophoresis</t> (PAGE) stained with Coomassie blue to localize the DT protein. Lane 1 shows the a DT-DBCO starting material ( M w , ~72,000), and lane 2 shows the a DT-siRNA conjugate ( M w , ~90,000) alone with some unreacted starting material. ( F ) Purification of the excess siRNA was confirmed via PAGE stained with GelRed to localize the siRNA. Lane 3 shows the a DT-siRNA conjugate along with unreacted siRNA; lane 4 shows the a DT-siRNA conjugate after nickel column purification, with only a small amount of excess siRNA left over.
    Polyacrylamide Gel Electrophoresis Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore trna sepharose column
    In vitro production of Arabidopsis <t>tRNA</t> ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA <t>agarose</t> as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).
    Trna Sepharose Column, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trna sepharose column/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trna sepharose column - by Bioz Stars, 2020-08
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    siRNAs can be conjugated to a DT. ( A ) a DT was engineered to contain a free cysteine as a functional handle ( a DT-SH), protected by a SUMO tag and purified using a histidine (His) tag. ( B ) a DT was reacted with a PEG cross-linker containing both maleimide and DBCO functional groups to obtain DBCO-modified a DT ( a DT-DBCO). ( C ) The presence of the DBCO modification on a DT was confirmed by measuring the absorbance at 280 nm ( a DT) and 309 nm (DBCO). Curves shown are a DT before modification (blue) and after DBCO modification (red). A 280 , absorbance at 280 nm. ( D ) Azide-modified siRNA was reacted with the DBCO-functionalized a DT to obtain the a DT-siRNA conjugate. RT, room temperature. ( E ) Modification of the a DT with the siRNA was confirmed via polyacrylamide gel electrophoresis (PAGE) stained with Coomassie blue to localize the DT protein. Lane 1 shows the a DT-DBCO starting material ( M w , ~72,000), and lane 2 shows the a DT-siRNA conjugate ( M w , ~90,000) alone with some unreacted starting material. ( F ) Purification of the excess siRNA was confirmed via PAGE stained with GelRed to localize the siRNA. Lane 3 shows the a DT-siRNA conjugate along with unreacted siRNA; lane 4 shows the a DT-siRNA conjugate after nickel column purification, with only a small amount of excess siRNA left over.

    Journal: Science Advances

    Article Title: Attenuated diphtheria toxin mediates siRNA delivery

    doi: 10.1126/sciadv.aaz4848

    Figure Lengend Snippet: siRNAs can be conjugated to a DT. ( A ) a DT was engineered to contain a free cysteine as a functional handle ( a DT-SH), protected by a SUMO tag and purified using a histidine (His) tag. ( B ) a DT was reacted with a PEG cross-linker containing both maleimide and DBCO functional groups to obtain DBCO-modified a DT ( a DT-DBCO). ( C ) The presence of the DBCO modification on a DT was confirmed by measuring the absorbance at 280 nm ( a DT) and 309 nm (DBCO). Curves shown are a DT before modification (blue) and after DBCO modification (red). A 280 , absorbance at 280 nm. ( D ) Azide-modified siRNA was reacted with the DBCO-functionalized a DT to obtain the a DT-siRNA conjugate. RT, room temperature. ( E ) Modification of the a DT with the siRNA was confirmed via polyacrylamide gel electrophoresis (PAGE) stained with Coomassie blue to localize the DT protein. Lane 1 shows the a DT-DBCO starting material ( M w , ~72,000), and lane 2 shows the a DT-siRNA conjugate ( M w , ~90,000) alone with some unreacted starting material. ( F ) Purification of the excess siRNA was confirmed via PAGE stained with GelRed to localize the siRNA. Lane 3 shows the a DT-siRNA conjugate along with unreacted siRNA; lane 4 shows the a DT-siRNA conjugate after nickel column purification, with only a small amount of excess siRNA left over.

    Article Snippet: Successful conjugation was confirmed by polyacrylamide gel electrophoresis analysis, and conjugation efficiency was quantified using ImageJ software (45% total yield; 80% recovery of DT, 55% siRNA conjugation efficiency).

    Techniques: Functional Assay, Purification, Modification, Polyacrylamide Gel Electrophoresis, Staining, Nickel Column

    In vitro production of Arabidopsis tRNA ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA agarose as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).

    Journal: Nucleic Acids Research

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    doi: 10.1093/nar/gki174

    Figure Lengend Snippet: In vitro production of Arabidopsis tRNA ligase and purification of the recombinant protein. Overexpression of the Arabidopsis tRNA ligase was accomplished in the RTS 100 wheat germ CECF system. Incubation of the vector DNA, carrying the tRNA ligase cDNA with a C-terminal histidine tag was performed in a 50 μl reaction mixture in the presence of [ 35 S]methionine for 24 h at 24°C. The overexpressed C-tagged tRNA ligase was subsequently purified by Ni-NTA chromatography. Aliquots of 2 μl of the total reaction mixture (lane a), of the flow-through (lane b) and wash fractions (lane c) after binding of the protein mixture to the Ni-NTA agarose as well as of the first fractions eluting with 500 mM imidazole (lanes d and e) were loaded onto a 7.5% polyacrylamide/0.1% SDS gel. After Coomassie brilliant blue staining (left panel), the gel was developed for fluorography (right panel) ( 61 ).

    Article Snippet: The pooled fractions from the tRNA Sepharose column were concentrated by ultrafiltration with Microcon 100 (Millipore, Schwalbach, Germany) and applied to a 10% polyacrylamide/0.1% SDS gel ( ).

    Techniques: In Vitro, Purification, Recombinant, Over Expression, Incubation, Plasmid Preparation, Chromatography, Flow Cytometry, Binding Assay, SDS-Gel, Staining

    Isolation of wheat germ tRNA ligase. ( A ) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. ( B ) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. ( C ) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc) 2 , 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10 4 c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.

    Journal: Nucleic Acids Research

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    doi: 10.1093/nar/gki174

    Figure Lengend Snippet: Isolation of wheat germ tRNA ligase. ( A ) Purification scheme. RNA ligase was purified from the soluble protein fraction of wheat embryos (S100 extract) by six consecutive steps. ( B ) As substrate for assaying tRNA ligase activity, we have used a natural modified Nicotiana pre-tRNA Tyr (NtY9-T7-M1). The arrows in the two 4 nt bulge loops indicate the 3′ and 5′ splice sites and dots identify the anticodon. ( C ) Fractionation of wheat germ tRNA ligase by Cibacron Blue Trisacryl M chromatography and ligation activity assay. Partially purified tRNA ligase from the Heparin Sepharose column was applied onto a Blue-Trisacryl M column. Elution of tRNA ligase was performed with a gradient of 150–800 mM KCl. Fractions of 5 ml were collected. Splicing endonuclease co-eluted with tRNA ligase at this purification step, generating 3′ and 5′ tRNA halves. Reaction mixtures (20 μl) contained 20 mM Tris–HCl, pH 7.5, 6 mM Mg(OAc) 2 , 80 μM spermine, 1 mM ATP, 0.5 mM GTP, 0.1 mM DTT, 0.5% Triton X-100, 40 fmol (4 × 10 4 c.p.m.) of T7-transcript (NtY9-T7-M1) and 2 μl from eluted fractions. Incubation was for 30 min at 37°C. Products were analysed on a 12.5% polyacrylamide/8 M urea gel. tRNA ligase activity elutes between 280 and 540 mM KCl as revealed by the detection of mature, spliced tRNA.

    Article Snippet: The pooled fractions from the tRNA Sepharose column were concentrated by ultrafiltration with Microcon 100 (Millipore, Schwalbach, Germany) and applied to a 10% polyacrylamide/0.1% SDS gel ( ).

    Techniques: Isolation, Purification, Activity Assay, Modification, Fractionation, Chromatography, Ligation, Incubation

    Gel filtration on Superdex™ 200 and adenylyltransferase activity of wheat germ tRNA ligase. ( A ) Partially purified tRNA ligase from the Source S15 column was subjected to gel filtration on Superdex™ 200. The column (HiLoad™ 16/60) was run with a flow rate of 1 ml/min. Fractions of 2 ml were collected. Aliquots of the elution fraction were analysed on a 7.5% polyacrylamide/0.1% SDS gel. The proteins were visualized by silver staining. ( B ) Appropriate aliquots of the indicated fractions were incubated in the presence of [α- 32 P]ATP for 15 min at 37°C. The ligase–[ 32 P]AMP adduct was detected by autoradiography of the dried gel. ( C ) The peak fractions from the tRNA Sepharose column were concentrated by ultrafiltration and 1/20 of this material was applied onto a 10% polyacrylamide/0.1% SDS gel and stained with Coomassie blue for analytical valuation. The arrows point to the position of the RNA ligase protein with an approximate molecular weight of 125 kDa. Protein size standards in kDa are indicated on the right.

    Journal: Nucleic Acids Research

    Article Title: Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    doi: 10.1093/nar/gki174

    Figure Lengend Snippet: Gel filtration on Superdex™ 200 and adenylyltransferase activity of wheat germ tRNA ligase. ( A ) Partially purified tRNA ligase from the Source S15 column was subjected to gel filtration on Superdex™ 200. The column (HiLoad™ 16/60) was run with a flow rate of 1 ml/min. Fractions of 2 ml were collected. Aliquots of the elution fraction were analysed on a 7.5% polyacrylamide/0.1% SDS gel. The proteins were visualized by silver staining. ( B ) Appropriate aliquots of the indicated fractions were incubated in the presence of [α- 32 P]ATP for 15 min at 37°C. The ligase–[ 32 P]AMP adduct was detected by autoradiography of the dried gel. ( C ) The peak fractions from the tRNA Sepharose column were concentrated by ultrafiltration and 1/20 of this material was applied onto a 10% polyacrylamide/0.1% SDS gel and stained with Coomassie blue for analytical valuation. The arrows point to the position of the RNA ligase protein with an approximate molecular weight of 125 kDa. Protein size standards in kDa are indicated on the right.

    Article Snippet: The pooled fractions from the tRNA Sepharose column were concentrated by ultrafiltration with Microcon 100 (Millipore, Schwalbach, Germany) and applied to a 10% polyacrylamide/0.1% SDS gel ( ).

    Techniques: Filtration, Activity Assay, Purification, Flow Cytometry, SDS-Gel, Silver Staining, Incubation, Autoradiography, Staining, Molecular Weight