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hela cell nuclear extracts in vitro transcription grade (Promega)

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Promega hela cell nuclear extracts in vitro transcription grade

<t>HeLa</t> cells were permeabilized with digitonin and washed in import buffer as described under “Experimental Procedures.” The reactions were carried out at 37 °C in transport buffer containing an ATP-regenerating system, 2 mm GTP, 5–7 mg/ml HeLa cytoplasmic extract, and 10 µg/ml Fl-PNA/pUSAG3. Reactions were stopped at 0.5 h (A), 1 h (B, 1.5 h (C), 2 h (D), 3 h (E), and 4 h (F) by washing the cells with wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. Half-micron sections of the cells were observed and digitized by confocal microscopy.

Hela Cell Nuclear Extracts In Vitro Transcription Grade, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hela+cell+nuclear+extract+in+vitro+transcription+grade/hela+cell+nuclear+extracts+in+vitro+transcription+grade/pmc04397984-55-19-28
Average 90 stars, based on 1 article reviews

hela cell nuclear extracts in vitro transcription grade - by Bioz Stars, 2026-09

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1) Product Images from "Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences * "

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

Journal: The Journal of biological chemistry

doi:

HeLa cells were permeabilized with digitonin and washed in import buffer as described under “Experimental Procedures.” The reactions were carried out at 37 °C in transport buffer containing an ATP-regenerating system, 2 mm GTP, 5–7 mg/ml HeLa cytoplasmic extract, and 10 µg/ml Fl-PNA/pUSAG3. Reactions were stopped at 0.5 h (A), 1 h (B, 1.5 h (C), 2 h (D), 3 h (E), and 4 h (F) by washing the cells with wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. Half-micron sections of the cells were observed and digitized by confocal microscopy.

Figure Legend Snippet: HeLa cells were permeabilized with digitonin and washed in import buffer as described under “Experimental Procedures.” The reactions were carried out at 37 °C in transport buffer containing an ATP-regenerating system, 2 mm GTP, 5–7 mg/ml HeLa cytoplasmic extract, and 10 µg/ml Fl-PNA/pUSAG3. Reactions were stopped at 0.5 h (A), 1 h (B, 1.5 h (C), 2 h (D), 3 h (E), and 4 h (F) by washing the cells with wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. Half-micron sections of the cells were observed and digitized by confocal microscopy.

Techniques Used: Confocal Microscopy

Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °C with either the Fl-PNA conjugated 4.2-kb plasmid pUSAG3 (A–D) or the Fl-PNA conjugated 14.4-kb plasmid pUSAG9 (E–H), both at 10 µg/ml, in the presence or absence of cellular extracts. BSA was added to 10 mg/ml to cells incubated in the absence of extracts (A and E). HeLa cell nuclear extract (Promega) was added to 0.5 mg/ml B and F). HeLa cell cytoplasmic extract was added to 5 mg/ml in the absence (C and G) or presence (D and H) of nuclear extract.

Figure Legend Snippet: Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °C with either the Fl-PNA conjugated 4.2-kb plasmid pUSAG3 (A–D) or the Fl-PNA conjugated 14.4-kb plasmid pUSAG9 (E–H), both at 10 µg/ml, in the presence or absence of cellular extracts. BSA was added to 10 mg/ml to cells incubated in the absence of extracts (A and E). HeLa cell nuclear extract (Promega) was added to 0.5 mg/ml B and F). HeLa cell cytoplasmic extract was added to 5 mg/ml in the absence (C and G) or presence (D and H) of nuclear extract.

Techniques Used: Incubation, Plasmid Preparation

Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °Cwith transport buffer lacking ATP, phosphocreatine, creatine phosphokinase, GTP, and Fl-PNA/pUSAG3 (10 µg/ml). The cells were then washed, fixed with 3% paraformaldehyde, and viewed by confocal microscopy. Cells incubated in the absence of cellular extracts contained 10 mg/ml BSA (A). Nuclear (B and D) and cytoplasmic (C and D) extracts were depleted of endogenous nucleotide triphosphates by preincubating the extracts with 10 units/ml apyrase at 25 °C for 30 min.

Figure Legend Snippet: Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °Cwith transport buffer lacking ATP, phosphocreatine, creatine phosphokinase, GTP, and Fl-PNA/pUSAG3 (10 µg/ml). The cells were then washed, fixed with 3% paraformaldehyde, and viewed by confocal microscopy. Cells incubated in the absence of cellular extracts contained 10 mg/ml BSA (A). Nuclear (B and D) and cytoplasmic (C and D) extracts were depleted of endogenous nucleotide triphosphates by preincubating the extracts with 10 units/ml apyrase at 25 °C for 30 min.

Techniques Used: Incubation, Confocal Microscopy

Permeabilized HeLa cells were incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml) for 4 h at 37 °C. All reactions contained HeLa cytoplasm at 5 mg/ml and either no additional lectin (A), concanavalin A (0.1 mg/ml; B) or wheat germ agglutinin (0.1 mg/ml; C).

Figure Legend Snippet: Permeabilized HeLa cells were incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml) for 4 h at 37 °C. All reactions contained HeLa cytoplasm at 5 mg/ml and either no additional lectin (A), concanavalin A (0.1 mg/ml; B) or wheat germ agglutinin (0.1 mg/ml; C).

Techniques Used: Incubation

HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSA-NLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. His-tagged importin α, importin β, and RAN were purified by nickel affinity chromatography and concentrated for addition to the reactions. The reactions contained BSA alone (10 mg/ml; A and E), nuclear extract alone (0.5 mg/ml; B and F), importin α, importin β, and RAN (0.5 mg/ml each; C and G), or importin α, importin β, RAN, and nuclear extract (D and H).

Figure Legend Snippet: HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSA-NLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. His-tagged importin α, importin β, and RAN were purified by nickel affinity chromatography and concentrated for addition to the reactions. The reactions contained BSA alone (10 mg/ml; A and E), nuclear extract alone (0.5 mg/ml; B and F), importin α, importin β, and RAN (0.5 mg/ml each; C and G), or importin α, importin β, RAN, and nuclear extract (D and H).

Techniques Used: Incubation, Purification, Affinity Chromatography

Figure Legend Snippet: HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSANLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. All reactions contained HeLa cell nuclear extract (0.5 mg/ml). Affinity purified His-tagged importins and RAN were added to 0.5 mg/ml each. The reactions contained importin α, importin β, and RAN (A and E), importin α, and RAN (B and F), importin β, and RAN (C and G) or importin α and importin β (D and H).

Techniques Used: Incubation, Affinity Purification

Permeabilized HeLa cells were incubated for 4 h at 37 °C with transport buffer containing HeLa cytoplasmic extract (5 mg/ml) and Fl-PNA/pUSAG3 (10 µg/ml) in the absence (A) or presence of a 1000-fold molar excess of BSA-NLS (0.23 mg/ml; B), a 100-fold excess of pUC19 (0.7 mg/ml; C), or a 100-fold molar excess of SV40 DNA (1.3 mg/ml; D).

Figure Legend Snippet: Permeabilized HeLa cells were incubated for 4 h at 37 °C with transport buffer containing HeLa cytoplasmic extract (5 mg/ml) and Fl-PNA/pUSAG3 (10 µg/ml) in the absence (A) or presence of a 1000-fold molar excess of BSA-NLS (0.23 mg/ml; B), a 100-fold excess of pUC19 (0.7 mg/ml; C), or a 100-fold molar excess of SV40 DNA (1.3 mg/ml; D).

Techniques Used: Incubation

Permeabilized HeLa cells were incubated for 4 h at 37 °C in transport buffer containing BSA alone (A and C) or HeLa cytoplasmic extract at 5 mg/ml (B and D). The substrates used were either Fl-PNA/pUSAG3 (A and B) or Fl-PNA/pUSAG3DSV40 (C and D), both present at 10 µg/ml.

Figure Legend Snippet: Permeabilized HeLa cells were incubated for 4 h at 37 °C in transport buffer containing BSA alone (A and C) or HeLa cytoplasmic extract at 5 mg/ml (B and D). The substrates used were either Fl-PNA/pUSAG3 (A and B) or Fl-PNA/pUSAG3DSV40 (C and D), both present at 10 µg/ml.

Techniques Used: Incubation

Plasmid DNA (0.1 µg) was incubated for 4 h at 37 °C with HeLa cell cytoplasmic extract (5 mg/ml) in transport buffer. Protein was removed by phenol: chloroform extraction, and the DNAs were separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed using a mixture of 32P-labeled pUSAG3 and pGenegrip blank. Lanes 1–3 contain 0.1 µg of unincubated plasmid used as starting material, and lanes 4–6 contain DNA incubated with cytoplasmic extract. Three plasmids were used: Fl-PNA/pUSAG3 (lanes 1 and 4), rhodamine-labeled PNA/ pGenegrip-GFP (lanes 2 and 5), and unlabeled pUSAG3 (lanes 3 and 6).

Figure Legend Snippet: Plasmid DNA (0.1 µg) was incubated for 4 h at 37 °C with HeLa cell cytoplasmic extract (5 mg/ml) in transport buffer. Protein was removed by phenol: chloroform extraction, and the DNAs were separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed using a mixture of 32P-labeled pUSAG3 and pGenegrip blank. Lanes 1–3 contain 0.1 µg of unincubated plasmid used as starting material, and lanes 4–6 contain DNA incubated with cytoplasmic extract. Three plasmids were used: Fl-PNA/pUSAG3 (lanes 1 and 4), rhodamine-labeled PNA/ pGenegrip-GFP (lanes 2 and 5), and unlabeled pUSAG3 (lanes 3 and 6).

Techniques Used: Plasmid Preparation, Incubation, Extraction, Agarose Gel Electrophoresis, Membrane, Labeling

In Vitro:

Article Title: A cis Element between the TATA Box and the Transcription Start Site of the Major Immediate-Early Promoter of Human Cytomegalovirus Determines Efficiency of Viral Replication
Article Snippet: .. Transcription mixtures, which contained approximately100 ng of template DNA, 200 g of HeLa cell nuclear extract from Promega (in vitro transcription grade), 3 mM MgCl2, and 10 Ci of [32P]UTP were as described previously (29). .. Reaction mixtures were incubated at 30°C for 20 min. After incubation, DNA templates were degraded with RNase-free DNase I (Promega) at 30°C for 30 min.

other:

Article Title: A cis Element between the TATA Box and the Transcription Start Site of the Major Immediate-Early Promoter of Human Cytomegalovirus Determines Efficiency of Viral Replication
Article Snippet: Reaction mixtures were incubated at 30°C for 20 min. After incubation, DNA templates were degraded with RNase-free DNase I (Promega) at 30°C for 30 min.

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Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: HeLa cells were permeabilized with digitonin and washed in import buffer as described under “Experimental Procedures.” The reactions were carried out at 37 °C in transport buffer containing an ATP-regenerating system, 2 mm GTP, 5–7 mg/ml HeLa cytoplasmic extract, and 10 µg/ml Fl-PNA/pUSAG3. Reactions were stopped at 0.5 h (A), 1 h (B, 1.5 h (C), 2 h (D), 3 h (E), and 4 h (F) by washing the cells with wash buffer and fixing them at 4 °C for 15 min in 3% paraformaldehyde in wash buffer. Half-micron sections of the cells were observed and digitized by confocal microscopy.

Article Snippet: Where indicated, cytoplasmic extracts prepared from HeLa cells ( 7 ) were added to between 5 and 7 mg/ml, HeLa cell nuclear extracts ( in vitro transcription grade, Promega, Madison, WI) were added to 0.25 mg/ml, and affinity purified, recombinant his-tagged RAN, importin α, and importin β were used at 0.5 mg/ml each.

Techniques: Confocal Microscopy

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °C with either the Fl-PNA conjugated 4.2-kb plasmid pUSAG3 (A–D) or the Fl-PNA conjugated 14.4-kb plasmid pUSAG9 (E–H), both at 10 µg/ml, in the presence or absence of cellular extracts. BSA was added to 10 mg/ml to cells incubated in the absence of extracts (A and E). HeLa cell nuclear extract (Promega) was added to 0.5 mg/ml B and F). HeLa cell cytoplasmic extract was added to 5 mg/ml in the absence (C and G) or presence (D and H) of nuclear extract.

Techniques: Incubation, Plasmid Preparation

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Digitonin permeabilized HeLa cells were incubated for 4 h at 37 °Cwith transport buffer lacking ATP, phosphocreatine, creatine phosphokinase, GTP, and Fl-PNA/pUSAG3 (10 µg/ml). The cells were then washed, fixed with 3% paraformaldehyde, and viewed by confocal microscopy. Cells incubated in the absence of cellular extracts contained 10 mg/ml BSA (A). Nuclear (B and D) and cytoplasmic (C and D) extracts were depleted of endogenous nucleotide triphosphates by preincubating the extracts with 10 units/ml apyrase at 25 °C for 30 min.

Techniques: Incubation, Confocal Microscopy

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Permeabilized HeLa cells were incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml) for 4 h at 37 °C. All reactions contained HeLa cytoplasm at 5 mg/ml and either no additional lectin (A), concanavalin A (0.1 mg/ml; B) or wheat germ agglutinin (0.1 mg/ml; C).

Techniques: Incubation

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSA-NLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. His-tagged importin α, importin β, and RAN were purified by nickel affinity chromatography and concentrated for addition to the reactions. The reactions contained BSA alone (10 mg/ml; A and E), nuclear extract alone (0.5 mg/ml; B and F), importin α, importin β, and RAN (0.5 mg/ml each; C and G), or importin α, importin β, RAN, and nuclear extract (D and H).

Techniques: Incubation, Purification, Affinity Chromatography

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: HeLa cells were permeabilized and incubated with transport buffer containing Fl-PNA/pUSAG3 (10 µg/ml; A–D) or Rh-BSA-NLS (25 µg/ml; E–H) at 37 °C with the indicated additions. Rh-BSANLS import reactions were incubated for 30 min, and pDNA import reactions proceeded for 4 h. All reactions contained HeLa cell nuclear extract (0.5 mg/ml). Affinity purified His-tagged importins and RAN were added to 0.5 mg/ml each. The reactions contained importin α, importin β, and RAN (A and E), importin α, and RAN (B and F), importin β, and RAN (C and G) or importin α and importin β (D and H).

Techniques: Incubation, Affinity Purification

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Permeabilized HeLa cells were incubated for 4 h at 37 °C with transport buffer containing HeLa cytoplasmic extract (5 mg/ml) and Fl-PNA/pUSAG3 (10 µg/ml) in the absence (A) or presence of a 1000-fold molar excess of BSA-NLS (0.23 mg/ml; B), a 100-fold excess of pUC19 (0.7 mg/ml; C), or a 100-fold molar excess of SV40 DNA (1.3 mg/ml; D).

Techniques: Incubation

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Permeabilized HeLa cells were incubated for 4 h at 37 °C in transport buffer containing BSA alone (A and C) or HeLa cytoplasmic extract at 5 mg/ml (B and D). The substrates used were either Fl-PNA/pUSAG3 (A and B) or Fl-PNA/pUSAG3DSV40 (C and D), both present at 10 µg/ml.

Techniques: Incubation

Journal: The Journal of biological chemistry

Article Title: Nuclear Import of Plasmid DNA in Digitonin-permeabilized Cells Requires Both Cytoplasmic Factors and Specific DNA Sequences *

doi:

Figure Lengend Snippet: Plasmid DNA (0.1 µg) was incubated for 4 h at 37 °C with HeLa cell cytoplasmic extract (5 mg/ml) in transport buffer. Protein was removed by phenol: chloroform extraction, and the DNAs were separated by agarose gel electrophoresis, transferred to a nylon membrane, and probed using a mixture of 32P-labeled pUSAG3 and pGenegrip blank. Lanes 1–3 contain 0.1 µg of unincubated plasmid used as starting material, and lanes 4–6 contain DNA incubated with cytoplasmic extract. Three plasmids were used: Fl-PNA/pUSAG3 (lanes 1 and 4), rhodamine-labeled PNA/ pGenegrip-GFP (lanes 2 and 5), and unlabeled pUSAG3 (lanes 3 and 6).

Techniques: Plasmid Preparation, Incubation, Extraction, Agarose Gel Electrophoresis, Membrane, Labeling

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hela cell nuclear extracts in vitro transcription grade (Promega)

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