Structured Review

Addgene inc vector plasmids pcdna3 bap sox2
Vector Plasmids Pcdna3 Bap Sox2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/vector plasmids pcdna3 bap sox2/product/Addgene inc
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
vector plasmids pcdna3 bap sox2 - by Bioz Stars, 2024-07
86/100 stars

Images


Structured Review

Addgene inc vector plasmids pcdna3 bap sox2
Validation of the interaction of BAP-X and BirA-Y. ( A ) Experimental workflow for the detection of protein–protein interactions between <t>SOX2</t> and OCT4. ( B ) Western blots of the experiment where X = SOX2 and Y = OCT4. The positions of BAP-fusions and nonspecific signals (NS) are indicated by asterisks. The biotin labeling time was 3 h. The BAP fragment has a His-tag, and the first western blot shows comparable total amounts of BAP-GFP and BAP-SOX2. The Streptavidin blot shows the number of biotin-labeled BAP-fusions which are the result of protein–protein interactions (or proximity) of BAP-SOX2 and BirA-OCT4 in lane 2. A very strong signal was observed in lane 2, and a very weak signal was observed in lane 1, which may be the result of a random collision of BAP-GFP and BirA-OCT4. Treatment with an anti-SOX2 antibody on the third blot indicates recombinant BAP-SOX2 and the absence of a detectable amount of endogenous SOX2. BAP adds a mass shift of 3 kDa, and only a single band was observed on the anti-SOX2 blot in lane 2. ( C ) Western blots of the reciprocal experiment where X = OCT4 and Y = SOX2. The biotin labeling time was 6 h. A comparable expression of BAP-GFP and BAP-OCT4 was detected on anti-His blot lanes 3 and 4. The left blots show the expression, the middle blots show the interaction and the right blots show the absence of the endogenous proteins SOX2 or OCT4. Note that BirA-X (which is actually BirA-8×His-tag-X) fusions were not observed on anti-His blots.
Vector Plasmids Pcdna3 Bap Sox2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/vector plasmids pcdna3 bap sox2/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
vector plasmids pcdna3 bap sox2 - by Bioz Stars, 2024-07
93/100 stars

Images

1) Product Images from "Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR"

Article Title: Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR

Journal: Life

doi: 10.3390/life13010107

Validation of the interaction of BAP-X and BirA-Y. ( A ) Experimental workflow for the detection of protein–protein interactions between SOX2 and OCT4. ( B ) Western blots of the experiment where X = SOX2 and Y = OCT4. The positions of BAP-fusions and nonspecific signals (NS) are indicated by asterisks. The biotin labeling time was 3 h. The BAP fragment has a His-tag, and the first western blot shows comparable total amounts of BAP-GFP and BAP-SOX2. The Streptavidin blot shows the number of biotin-labeled BAP-fusions which are the result of protein–protein interactions (or proximity) of BAP-SOX2 and BirA-OCT4 in lane 2. A very strong signal was observed in lane 2, and a very weak signal was observed in lane 1, which may be the result of a random collision of BAP-GFP and BirA-OCT4. Treatment with an anti-SOX2 antibody on the third blot indicates recombinant BAP-SOX2 and the absence of a detectable amount of endogenous SOX2. BAP adds a mass shift of 3 kDa, and only a single band was observed on the anti-SOX2 blot in lane 2. ( C ) Western blots of the reciprocal experiment where X = OCT4 and Y = SOX2. The biotin labeling time was 6 h. A comparable expression of BAP-GFP and BAP-OCT4 was detected on anti-His blot lanes 3 and 4. The left blots show the expression, the middle blots show the interaction and the right blots show the absence of the endogenous proteins SOX2 or OCT4. Note that BirA-X (which is actually BirA-8×His-tag-X) fusions were not observed on anti-His blots.
Figure Legend Snippet: Validation of the interaction of BAP-X and BirA-Y. ( A ) Experimental workflow for the detection of protein–protein interactions between SOX2 and OCT4. ( B ) Western blots of the experiment where X = SOX2 and Y = OCT4. The positions of BAP-fusions and nonspecific signals (NS) are indicated by asterisks. The biotin labeling time was 3 h. The BAP fragment has a His-tag, and the first western blot shows comparable total amounts of BAP-GFP and BAP-SOX2. The Streptavidin blot shows the number of biotin-labeled BAP-fusions which are the result of protein–protein interactions (or proximity) of BAP-SOX2 and BirA-OCT4 in lane 2. A very strong signal was observed in lane 2, and a very weak signal was observed in lane 1, which may be the result of a random collision of BAP-GFP and BirA-OCT4. Treatment with an anti-SOX2 antibody on the third blot indicates recombinant BAP-SOX2 and the absence of a detectable amount of endogenous SOX2. BAP adds a mass shift of 3 kDa, and only a single band was observed on the anti-SOX2 blot in lane 2. ( C ) Western blots of the reciprocal experiment where X = OCT4 and Y = SOX2. The biotin labeling time was 6 h. A comparable expression of BAP-GFP and BAP-OCT4 was detected on anti-His blot lanes 3 and 4. The left blots show the expression, the middle blots show the interaction and the right blots show the absence of the endogenous proteins SOX2 or OCT4. Note that BirA-X (which is actually BirA-8×His-tag-X) fusions were not observed on anti-His blots.

Techniques Used: Western Blot, Labeling, Recombinant, Expressing

Real-time qRT-PCR amplification of OCT4 and SOX2 in the PUB experiments. Error bars represent the standard deviation.
Figure Legend Snippet: Real-time qRT-PCR amplification of OCT4 and SOX2 in the PUB experiments. Error bars represent the standard deviation.

Techniques Used: Quantitative RT-PCR, Amplification, Standard Deviation


Structured Review

Addgene inc vector plasmids pcdna3 bap sox2
Experimental workflow for the detection and quantitative analysis of protein–protein interactions (PPI) between pluripotency transcription factors. Cells expressing biotin acceptor peptide <t>(BAP)-Sox2</t> and BirA-Oct4 were labelled by biotin (3 h or 9 h) before harvest. The cells were lysed in cytoskeleton (CSK) buffer with 0.5% Triton X-100 to isolate the nuclei. The nuclear fractionsere incubated on Ni-sepharose beads to purify and enrich the fused proteins. The Ni-sepharose-bound proteins were treated by propionic anhydride, washed, and digested on the beads with trypsin, and the tryptic peptides were desalted by Ziptips. The peptide mixtures were then analyzed by LC–MS/MS. The propionylated and biotinylated BAP peptides were identified and quantified using Data Analysis software.
Vector Plasmids Pcdna3 Bap Sox2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/vector plasmids pcdna3 bap sox2/product/Addgene inc
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
vector plasmids pcdna3 bap sox2 - by Bioz Stars, 2024-07
93/100 stars

Images

1) Product Images from "In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation"

Article Title: In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Journal: Biomolecules

doi: 10.3390/biom10010142

Experimental workflow for the detection and quantitative analysis of protein–protein interactions (PPI) between pluripotency transcription factors. Cells expressing biotin acceptor peptide (BAP)-Sox2 and BirA-Oct4 were labelled by biotin (3 h or 9 h) before harvest. The cells were lysed in cytoskeleton (CSK) buffer with 0.5% Triton X-100 to isolate the nuclei. The nuclear fractionsere incubated on Ni-sepharose beads to purify and enrich the fused proteins. The Ni-sepharose-bound proteins were treated by propionic anhydride, washed, and digested on the beads with trypsin, and the tryptic peptides were desalted by Ziptips. The peptide mixtures were then analyzed by LC–MS/MS. The propionylated and biotinylated BAP peptides were identified and quantified using Data Analysis software.
Figure Legend Snippet: Experimental workflow for the detection and quantitative analysis of protein–protein interactions (PPI) between pluripotency transcription factors. Cells expressing biotin acceptor peptide (BAP)-Sox2 and BirA-Oct4 were labelled by biotin (3 h or 9 h) before harvest. The cells were lysed in cytoskeleton (CSK) buffer with 0.5% Triton X-100 to isolate the nuclei. The nuclear fractionsere incubated on Ni-sepharose beads to purify and enrich the fused proteins. The Ni-sepharose-bound proteins were treated by propionic anhydride, washed, and digested on the beads with trypsin, and the tryptic peptides were desalted by Ziptips. The peptide mixtures were then analyzed by LC–MS/MS. The propionylated and biotinylated BAP peptides were identified and quantified using Data Analysis software.

Techniques Used: Expressing, Incubation, Liquid Chromatography with Mass Spectroscopy, Software

( a ) Biotinylationis interaction/proximity-dependent (DNA-dependent binary interaction between Sox2 and Oct4). The positions of the BAPfusion proteins and nonspecific signal (NS) are indicated. Importantly, the NS was also detected in untransfected cells and corresponds to cellular proteins that bind to the anti-His antibodies. Expression of BAP-GFP was used as a control (since GFP does not have a DNA-binding domain). Western blot using anti-His-HRP shows the total expression levels of the recombinant proteins BAP-GFP, BAP-Sox2, and BAP-Oct4 (Top, lanes 1–4). Strong signals in the streptavidin-HRP Western blot identifyproteins which are in proximity in vivo (lanes 3, 4, Bottom). Biotin pulse, 30 min. ( b ) Since the high-mobility group (HMG) domain of BAP-Sox2 and the POU domain of BirA-Oct4 assemble on closely spaced composites of CATTGTC-like and ATGCAAAT-like DNA binding sites, the BAP target will be biotinylated. No biotinylation was observed when coexpressing BAP-GFP and BirA-Sox2 and BAP-GFP and BirA-Oct4 (lanes 1 and 2).
Figure Legend Snippet: ( a ) Biotinylationis interaction/proximity-dependent (DNA-dependent binary interaction between Sox2 and Oct4). The positions of the BAPfusion proteins and nonspecific signal (NS) are indicated. Importantly, the NS was also detected in untransfected cells and corresponds to cellular proteins that bind to the anti-His antibodies. Expression of BAP-GFP was used as a control (since GFP does not have a DNA-binding domain). Western blot using anti-His-HRP shows the total expression levels of the recombinant proteins BAP-GFP, BAP-Sox2, and BAP-Oct4 (Top, lanes 1–4). Strong signals in the streptavidin-HRP Western blot identifyproteins which are in proximity in vivo (lanes 3, 4, Bottom). Biotin pulse, 30 min. ( b ) Since the high-mobility group (HMG) domain of BAP-Sox2 and the POU domain of BirA-Oct4 assemble on closely spaced composites of CATTGTC-like and ATGCAAAT-like DNA binding sites, the BAP target will be biotinylated. No biotinylation was observed when coexpressing BAP-GFP and BirA-Sox2 and BAP-GFP and BirA-Oct4 (lanes 1 and 2).

Techniques Used: Expressing, Binding Assay, Western Blot, Recombinant, In Vivo

The biotinylation levels are interaction/proximity-dependent. ( a ) Western blotting to detect the interaction of pluripotency transcription factors versus a control at two biotin pulse times. Combinations of BirA and BAP fusion proteins were transfected separately into cells: lanes 1 and 3: BAP-GFP + BirA-Oct4 (control), lanes 2 and 4: BAP-Sox2 + BirA-Oct4, 0: nontransfected cells, NS: nonspecific signal. ( b ) Multiple reaction monitoring (MRM) detection of propionylated and biotinylated BAPs. Shown are extracted ions chromatograms for the most intensive fragmentation ions present in the MS/MS spectra of the respective peptides (y-series). Note the change of peaks corresponding to biotinylated BAP (colored blue) relative to propionylated BAP (colored brown) at different biotin pulse times (sample 2 and sample 4). Numbers in the graphs correspond to the lane numbers in the Western blots.
Figure Legend Snippet: The biotinylation levels are interaction/proximity-dependent. ( a ) Western blotting to detect the interaction of pluripotency transcription factors versus a control at two biotin pulse times. Combinations of BirA and BAP fusion proteins were transfected separately into cells: lanes 1 and 3: BAP-GFP + BirA-Oct4 (control), lanes 2 and 4: BAP-Sox2 + BirA-Oct4, 0: nontransfected cells, NS: nonspecific signal. ( b ) Multiple reaction monitoring (MRM) detection of propionylated and biotinylated BAPs. Shown are extracted ions chromatograms for the most intensive fragmentation ions present in the MS/MS spectra of the respective peptides (y-series). Note the change of peaks corresponding to biotinylated BAP (colored blue) relative to propionylated BAP (colored brown) at different biotin pulse times (sample 2 and sample 4). Numbers in the graphs correspond to the lane numbers in the Western blots.

Techniques Used: Western Blot, Transfection, Tandem Mass Spectroscopy

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Addgene inc vector plasmids pcdna3 bap sox2
    Vector Plasmids Pcdna3 Bap Sox2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vector plasmids pcdna3 bap sox2/product/Addgene inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vector plasmids pcdna3 bap sox2 - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    Image Search Results