protein g beads  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Name:
    Dynabeads Protein G Immunoprecipitation Kit
    Description:
    The Dynabeads Protein G Immunoprecipitation Kit is a faster and easier solution for immunoprecipitation IP than using Sepharose resin or agarose resin and includes all reagents and buffers required to perform IP using your own antibody The kit is optimized for standard IP but can also be used for Co IP chromatin IP ChIP or RNA IP RIP The kit includes the widely used Dynabeads Protein G with recombinant Protein G 17 kDa covalently coupled to the magnetic bead surface Both manual and automated protocols are available • IP in less than 40 minutes • High target protein yield with low antibody consumption • Very low non specific binding with high signal to noise ratio • No columns centrifugations or time consuming pre clearing required • High reproducibility with Dynabeads Protein G and all buffers supplied in the kit • High throughput compatible with KingFisher instruments Manual Dynabeads separation is fast and easy to perform The manual protocol is simple and can be performed in under 40 minutes First the antibody for the target protein is incubated with the Dynabeads Protein G in a tube for 10 minutes Excess antibody is washed away by placing the tube in a DynaMag magnet and removing the supernatant The antibody coated beads can then be used for a variety of downstream applications including IP Co IP chromatin IP ChIP RNA IP RIP small scale IgG purification and protein purification Bound material is easily collected using a DynaMag magnet due to the unique magnetic properties of the Dynabeads The recombinant protein G on the beads contains no albumin binding sites thus albumin is not co purified during the procedure The IP is fast and gives high yield high reproducibility and very little non specific binding thus pre clearing is not required Automated Dynabeads separation helps increase throughput and reduces hands on time If you are working with several samples in parallel the number of washing steps and the hands on time increases proportionally with the number of samples Pipetting and other manual handling tend to be less consistent than automation when working with many samples at a time To better handle a medium to high throughput number of samples reduce hands on time and secure high reproducibility we have developed IP protocols for the KingFisher Flex and KingFisher Duo Prime instruments The automated protocols replicate the manual protocols obtaining equally high target protein yield and the same low non specific binding and high reproducibility It doesn t matter if you are working with 10 or 96 samples the IP protocol is less than 40 minutes regardless Just load the reagents on the plates push the “Start button and by the time you have prepared for downstream analysis the IP is done Some optimization e g incubation times might be necessary depending on your antibody and the abundance and or specificity of your target protein • Use the KingFisher Duo instrument for low to medium throughput 1 12 samples run • Use the KingFisher Flex instrument for high throughput 12 96 samples run See automated protocols Watch a video about the KingFisher Flex instrument Gentle separation causes minimal physical stress to proteins The magnetic separation technology utilized by Dynabeads Protein G is rapid and gentle causing minimal physical stress to your target proteins This permits the isolation and concentration of labile composites that might otherwise dissociate or be damaged by proteases during long incubation times Native protein conformation and large protein complexes are preserved Binding strength and capacity Dynabeads Protein G allow for isolation of most mammalian immunoglobulins Ig The amount of Ig captured depends on the concentration of Ig in the starting sample and on the type and source of the Ig 100 µL of Dynabeads Protein G will isolate approximately 25 30 µg human IgG from a sample containing 20 200 µg IgG mL Predominant Fc binding allows optimal Ig orientation The antibodies bind to the outer smooth surface of the beads thus are not trapped in large pores as with Sepharose agarose based beads All antibodies are available for protein binding so low amounts of antibody are required while still obtaining the same high yield of target protein The smooth bead surface is also responsible for the low non specific binding that Dynabeads are known for Learn more about Dynabeads • Dynabeads Protein G are also available as a stand alone reagent without included buffers • See immunoprecipitation selection guides data and references • See magnets for Dynabeads separations • Find Dynabeads products for other applications OEM purchase To purchase Dynabeads Protein A and Protein G on an OEM basis contact our Out Licensing and OEM Sales department Sepharose is a trademark of GE Healthcare Bio Sciences AB
    Catalog Number:
    10007d
    Price:
    None
    Applications:
    Cell Lysis & Fractionation|Immunoprecipitation|Organelle Isolation|Protein Assays and Analysis|Protein Biology|Protein Purification & Isolation
    Category:
    Beads Microspheres
    Buy from Supplier


    Structured Review

    Thermo Fisher protein g beads
    β-1,3-glucan is required for NKp30-mediated killing of Cryptococcus . a Flow cytometric analysis of cryptococcal cell wall/membrane (CCW/M) binding to YT cells as detected by mAb to β-1,3-glucan vs. isotype control Ab. b Flow cytometric analysis of β-glucan binding to YT cell. YT cells were incubated with (left panel) or without (right panel) laminarin, a linear β-1,3-glucan with β-1,6-linkages. c Flow cytometric analysis of laminarihexaose binding to YT cells. YT cells were incubated with laminarihexaose (from curdlan, comprising only β-1,3-glucan). d Comparison of binding of pustulan vs. β-1,3-glucan to YT cells analyzed using flow cytometry. YT cells were incubated with pustulan (comprising only β-1,6-glucan) or β-1,3-glucan (derived from S. cerevisiae ). This experiment was performed twice. e Flow cytometric analysis of a recombinant NKp30-Fc fusion protein binding to β-glucan-conjugated beads (β-1,3-GB) compared to unconjugated polystyrene beads as control. NKp30-Fc on the beads was detected with anti-NKp30 antibody (1C01). f Anti-NKp30 mAb (1C01) blocked NKp30 binding to β-1,3-GB. Recombinant NKp30 was incubated with 1C01 before being applied to beads conjugated with β-1,3-glucan. The presence of NKp30 on β-1,3-GB was detected using the polyclonal anti-NKp30 antibody. g NKp30 binding to C. neoformans vs. C. albicans analyzed using flow cytometry. The experiment was performed twice. h Immunoprecipitation of NKp30 with β-1,3-glucan. YT cell lysate was incubated with β-1,3-glucan (laminarin) before being incubated with <t>protein</t> G beads that had been conjugated with a mAb against β-1,3-glucan. i YT cell killing of C. neoformans ). **, p
    The Dynabeads Protein G Immunoprecipitation Kit is a faster and easier solution for immunoprecipitation IP than using Sepharose resin or agarose resin and includes all reagents and buffers required to perform IP using your own antibody The kit is optimized for standard IP but can also be used for Co IP chromatin IP ChIP or RNA IP RIP The kit includes the widely used Dynabeads Protein G with recombinant Protein G 17 kDa covalently coupled to the magnetic bead surface Both manual and automated protocols are available • IP in less than 40 minutes • High target protein yield with low antibody consumption • Very low non specific binding with high signal to noise ratio • No columns centrifugations or time consuming pre clearing required • High reproducibility with Dynabeads Protein G and all buffers supplied in the kit • High throughput compatible with KingFisher instruments Manual Dynabeads separation is fast and easy to perform The manual protocol is simple and can be performed in under 40 minutes First the antibody for the target protein is incubated with the Dynabeads Protein G in a tube for 10 minutes Excess antibody is washed away by placing the tube in a DynaMag magnet and removing the supernatant The antibody coated beads can then be used for a variety of downstream applications including IP Co IP chromatin IP ChIP RNA IP RIP small scale IgG purification and protein purification Bound material is easily collected using a DynaMag magnet due to the unique magnetic properties of the Dynabeads The recombinant protein G on the beads contains no albumin binding sites thus albumin is not co purified during the procedure The IP is fast and gives high yield high reproducibility and very little non specific binding thus pre clearing is not required Automated Dynabeads separation helps increase throughput and reduces hands on time If you are working with several samples in parallel the number of washing steps and the hands on time increases proportionally with the number of samples Pipetting and other manual handling tend to be less consistent than automation when working with many samples at a time To better handle a medium to high throughput number of samples reduce hands on time and secure high reproducibility we have developed IP protocols for the KingFisher Flex and KingFisher Duo Prime instruments The automated protocols replicate the manual protocols obtaining equally high target protein yield and the same low non specific binding and high reproducibility It doesn t matter if you are working with 10 or 96 samples the IP protocol is less than 40 minutes regardless Just load the reagents on the plates push the “Start button and by the time you have prepared for downstream analysis the IP is done Some optimization e g incubation times might be necessary depending on your antibody and the abundance and or specificity of your target protein • Use the KingFisher Duo instrument for low to medium throughput 1 12 samples run • Use the KingFisher Flex instrument for high throughput 12 96 samples run See automated protocols Watch a video about the KingFisher Flex instrument Gentle separation causes minimal physical stress to proteins The magnetic separation technology utilized by Dynabeads Protein G is rapid and gentle causing minimal physical stress to your target proteins This permits the isolation and concentration of labile composites that might otherwise dissociate or be damaged by proteases during long incubation times Native protein conformation and large protein complexes are preserved Binding strength and capacity Dynabeads Protein G allow for isolation of most mammalian immunoglobulins Ig The amount of Ig captured depends on the concentration of Ig in the starting sample and on the type and source of the Ig 100 µL of Dynabeads Protein G will isolate approximately 25 30 µg human IgG from a sample containing 20 200 µg IgG mL Predominant Fc binding allows optimal Ig orientation The antibodies bind to the outer smooth surface of the beads thus are not trapped in large pores as with Sepharose agarose based beads All antibodies are available for protein binding so low amounts of antibody are required while still obtaining the same high yield of target protein The smooth bead surface is also responsible for the low non specific binding that Dynabeads are known for Learn more about Dynabeads • Dynabeads Protein G are also available as a stand alone reagent without included buffers • See immunoprecipitation selection guides data and references • See magnets for Dynabeads separations • Find Dynabeads products for other applications OEM purchase To purchase Dynabeads Protein A and Protein G on an OEM basis contact our Out Licensing and OEM Sales department Sepharose is a trademark of GE Healthcare Bio Sciences AB
    https://www.bioz.com/result/protein g beads/product/Thermo Fisher
    Average 90 stars, based on 2596 article reviews
    Price from $9.99 to $1999.99
    protein g beads - by Bioz Stars, 2020-02
    90/100 stars

    Images

    1) Product Images from "Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida"

    Article Title: Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03014-4

    β-1,3-glucan is required for NKp30-mediated killing of Cryptococcus . a Flow cytometric analysis of cryptococcal cell wall/membrane (CCW/M) binding to YT cells as detected by mAb to β-1,3-glucan vs. isotype control Ab. b Flow cytometric analysis of β-glucan binding to YT cell. YT cells were incubated with (left panel) or without (right panel) laminarin, a linear β-1,3-glucan with β-1,6-linkages. c Flow cytometric analysis of laminarihexaose binding to YT cells. YT cells were incubated with laminarihexaose (from curdlan, comprising only β-1,3-glucan). d Comparison of binding of pustulan vs. β-1,3-glucan to YT cells analyzed using flow cytometry. YT cells were incubated with pustulan (comprising only β-1,6-glucan) or β-1,3-glucan (derived from S. cerevisiae ). This experiment was performed twice. e Flow cytometric analysis of a recombinant NKp30-Fc fusion protein binding to β-glucan-conjugated beads (β-1,3-GB) compared to unconjugated polystyrene beads as control. NKp30-Fc on the beads was detected with anti-NKp30 antibody (1C01). f Anti-NKp30 mAb (1C01) blocked NKp30 binding to β-1,3-GB. Recombinant NKp30 was incubated with 1C01 before being applied to beads conjugated with β-1,3-glucan. The presence of NKp30 on β-1,3-GB was detected using the polyclonal anti-NKp30 antibody. g NKp30 binding to C. neoformans vs. C. albicans analyzed using flow cytometry. The experiment was performed twice. h Immunoprecipitation of NKp30 with β-1,3-glucan. YT cell lysate was incubated with β-1,3-glucan (laminarin) before being incubated with protein G beads that had been conjugated with a mAb against β-1,3-glucan. i YT cell killing of C. neoformans ). **, p
    Figure Legend Snippet: β-1,3-glucan is required for NKp30-mediated killing of Cryptococcus . a Flow cytometric analysis of cryptococcal cell wall/membrane (CCW/M) binding to YT cells as detected by mAb to β-1,3-glucan vs. isotype control Ab. b Flow cytometric analysis of β-glucan binding to YT cell. YT cells were incubated with (left panel) or without (right panel) laminarin, a linear β-1,3-glucan with β-1,6-linkages. c Flow cytometric analysis of laminarihexaose binding to YT cells. YT cells were incubated with laminarihexaose (from curdlan, comprising only β-1,3-glucan). d Comparison of binding of pustulan vs. β-1,3-glucan to YT cells analyzed using flow cytometry. YT cells were incubated with pustulan (comprising only β-1,6-glucan) or β-1,3-glucan (derived from S. cerevisiae ). This experiment was performed twice. e Flow cytometric analysis of a recombinant NKp30-Fc fusion protein binding to β-glucan-conjugated beads (β-1,3-GB) compared to unconjugated polystyrene beads as control. NKp30-Fc on the beads was detected with anti-NKp30 antibody (1C01). f Anti-NKp30 mAb (1C01) blocked NKp30 binding to β-1,3-GB. Recombinant NKp30 was incubated with 1C01 before being applied to beads conjugated with β-1,3-glucan. The presence of NKp30 on β-1,3-GB was detected using the polyclonal anti-NKp30 antibody. g NKp30 binding to C. neoformans vs. C. albicans analyzed using flow cytometry. The experiment was performed twice. h Immunoprecipitation of NKp30 with β-1,3-glucan. YT cell lysate was incubated with β-1,3-glucan (laminarin) before being incubated with protein G beads that had been conjugated with a mAb against β-1,3-glucan. i YT cell killing of C. neoformans ). **, p

    Techniques Used: Flow Cytometry, Binding Assay, Incubation, Cytometry, Derivative Assay, Recombinant, Protein Binding, Immunoprecipitation

    2) Product Images from "Adipocyte nuclei captured from VAT and SAT"

    Article Title: Adipocyte nuclei captured from VAT and SAT

    Journal: BMC obesity

    doi: 10.1186/s40608-016-0112-6

    Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with Protein-G Dynabeads (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei
    Figure Legend Snippet: Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with Protein-G Dynabeads (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei

    Techniques Used: Expressing, Fluorescence, Plasmid Preparation, Staining, Negative Control

    Mature MA-INTACT mouse adipocyte nuclei expressed SUN1mRFP1Flag RNA in BAT, VAT, and SAT allowing adipocyte nuclei to be captured on Immuno-paramagnetic beads. a Illustration of mouse fat deposits examined. b , c , d , e . Nuclei were efficiently captured from crude nuclear preparations prepared from inguinal SAT ( b ) retroperitioneal VAT ( c ) epididymal VAT ( d ) and scapular BAT ( e ). Protein G Dynabeads pre-adsorbed to rabbit anti-mRFP1 polyclonal antibody were used in these capture experiments. The clumping of nuclei occurs during successive rounds of washing and capture. Negative control antibodies did not capture significant numbers of nuclei (not shown). f A Western blot ( top panel ) showed that preparations of captured VAT, SAT, and BAT nuclei expressed the 131 kDa SUN1mRFP1Flag reporter protein, while uncaptured nuclei did not. In house prepared anti-mRFP1 rabbit pAb detected the mRFP1 domain. Purified 25 kDa mRFP1 was run as a positive control. A loading control ( bottom panel ) showed the approximately equal loading of nuclear proteins and an mRFP1 standard. The loading control samples were run for a short time (20 min) on an SDS PAGE system and protein front stained with Coomassie
    Figure Legend Snippet: Mature MA-INTACT mouse adipocyte nuclei expressed SUN1mRFP1Flag RNA in BAT, VAT, and SAT allowing adipocyte nuclei to be captured on Immuno-paramagnetic beads. a Illustration of mouse fat deposits examined. b , c , d , e . Nuclei were efficiently captured from crude nuclear preparations prepared from inguinal SAT ( b ) retroperitioneal VAT ( c ) epididymal VAT ( d ) and scapular BAT ( e ). Protein G Dynabeads pre-adsorbed to rabbit anti-mRFP1 polyclonal antibody were used in these capture experiments. The clumping of nuclei occurs during successive rounds of washing and capture. Negative control antibodies did not capture significant numbers of nuclei (not shown). f A Western blot ( top panel ) showed that preparations of captured VAT, SAT, and BAT nuclei expressed the 131 kDa SUN1mRFP1Flag reporter protein, while uncaptured nuclei did not. In house prepared anti-mRFP1 rabbit pAb detected the mRFP1 domain. Purified 25 kDa mRFP1 was run as a positive control. A loading control ( bottom panel ) showed the approximately equal loading of nuclear proteins and an mRFP1 standard. The loading control samples were run for a short time (20 min) on an SDS PAGE system and protein front stained with Coomassie

    Techniques Used: Negative Control, Western Blot, Purification, Positive Control, SDS Page, Staining

    3) Product Images from "Post-transcriptional gene silencing mediated by microRNAs is controlled by nucleoplasmic Sfpq"

    Article Title: Post-transcriptional gene silencing mediated by microRNAs is controlled by nucleoplasmic Sfpq

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01126-x

    Sfpq, Pspc1, and NonO are components of Ago2 complex and interact with let-7a. a Overview of the proteomic method used to identify RNA-dependent proteins interacting with Ago2. b Silver staining of an SDS-PAGE analysis from the IP with the anti-Ago2 antibody, the protein G conjugated with the Dynabeads (DB), or the anti-Ago2 antibody alone incubated without cell lysate. The samples were untreated, treated with 10 μg ml −1 RNase A for 30 min at room temperature (for partial digestion), or treated with 10 mg ml −1 RNase A for 30 min at room temperature (for total digestion). c Radioactive images of a TBE-Urea gel showing signal from 32 P-labeled RNA fragments of samples untreated, treated with 10 μg ml −1 RNase A for 30 min at room temperature (for partial digestion), or treated with 10 mg ml −1 RNase A for 30 min at room temperature (for total digestion). d Scatter-plot of the log base 2 (−/+) RNase A ratios (abundance scores) plotted with the unique peptides for each identified protein. Each spot is a different protein. e Co-IP of endogenous Sfpq and Ago2, Pspc1, or NonO in RAW 264.7 and HEK293T cells. When indicated, cell lysates were incubated at room temperature with RNase A (10 mg ml −1 ) for 30 min. f Sfpq and Pspc1 interact with mature let-7a but not with the precursor. RAW 264.7 cell extracts were immunoprecipitated with two different antibodies for each indicated protein. The RNA was purified and analyzed by Northern blotting
    Figure Legend Snippet: Sfpq, Pspc1, and NonO are components of Ago2 complex and interact with let-7a. a Overview of the proteomic method used to identify RNA-dependent proteins interacting with Ago2. b Silver staining of an SDS-PAGE analysis from the IP with the anti-Ago2 antibody, the protein G conjugated with the Dynabeads (DB), or the anti-Ago2 antibody alone incubated without cell lysate. The samples were untreated, treated with 10 μg ml −1 RNase A for 30 min at room temperature (for partial digestion), or treated with 10 mg ml −1 RNase A for 30 min at room temperature (for total digestion). c Radioactive images of a TBE-Urea gel showing signal from 32 P-labeled RNA fragments of samples untreated, treated with 10 μg ml −1 RNase A for 30 min at room temperature (for partial digestion), or treated with 10 mg ml −1 RNase A for 30 min at room temperature (for total digestion). d Scatter-plot of the log base 2 (−/+) RNase A ratios (abundance scores) plotted with the unique peptides for each identified protein. Each spot is a different protein. e Co-IP of endogenous Sfpq and Ago2, Pspc1, or NonO in RAW 264.7 and HEK293T cells. When indicated, cell lysates were incubated at room temperature with RNase A (10 mg ml −1 ) for 30 min. f Sfpq and Pspc1 interact with mature let-7a but not with the precursor. RAW 264.7 cell extracts were immunoprecipitated with two different antibodies for each indicated protein. The RNA was purified and analyzed by Northern blotting

    Techniques Used: Silver Staining, SDS Page, Incubation, Labeling, Co-Immunoprecipitation Assay, Immunoprecipitation, Purification, Northern Blot

    4) Product Images from "The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus"

    Article Title: The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03347

    YpkA and SakA interact in vivo during heat shock stress. The wild-type and sakA ::GFP ypkA ::3xHA strains were used in the Co-IP assays. Strains were grown at 30°C (24 h) and subsequently exposed to heat shock at 48°C for the indicated times. (A) GFP-Trap resin was used to immunoprecipitate SakA::GFP. (B) Dynabeads Protein A were incubated with monoclonal α-HA antibody and used to immunoprecipitate YpkA::3xHA in reciprocal experiment. Co-immunoprecipitated proteins were investigated via Western blot analysis using α-HA and α-GFP antibodies. The Coomassie Brilliant Blue (CBB) stained gel was used as an additional loading sample control.
    Figure Legend Snippet: YpkA and SakA interact in vivo during heat shock stress. The wild-type and sakA ::GFP ypkA ::3xHA strains were used in the Co-IP assays. Strains were grown at 30°C (24 h) and subsequently exposed to heat shock at 48°C for the indicated times. (A) GFP-Trap resin was used to immunoprecipitate SakA::GFP. (B) Dynabeads Protein A were incubated with monoclonal α-HA antibody and used to immunoprecipitate YpkA::3xHA in reciprocal experiment. Co-immunoprecipitated proteins were investigated via Western blot analysis using α-HA and α-GFP antibodies. The Coomassie Brilliant Blue (CBB) stained gel was used as an additional loading sample control.

    Techniques Used: In Vivo, Co-Immunoprecipitation Assay, Incubation, Immunoprecipitation, Western Blot, Staining

    5) Product Images from "Two E3 ligases antagonistically regulate the UV-B response in Arabidopsis"

    Article Title: Two E3 ligases antagonistically regulate the UV-B response in Arabidopsis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1816268116

    COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.
    Figure Legend Snippet: COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.

    Techniques Used: Negative Control, In Vitro, Purification, Incubation, Transfection, Co-Immunoprecipitation Assay, In Vivo, Magnetic Beads

    6) Product Images from "PAI1 blocks NMDA receptor-mediated effects of tissue-type plasminogen activator on cell signaling and physiology"

    Article Title: PAI1 blocks NMDA receptor-mediated effects of tissue-type plasminogen activator on cell signaling and physiology

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.217083

    PAI1 inhibits binding of EA-tPA to immunopurified NMDA-R. (A) Extracts of mouse cortex were incubated with NMDA-R NR1 subunit-specific antibody (NR1) or with NS-IgG (IgG) coupled to Protein-G–Dynabeads. Bound proteins were immunoprecipitated in
    Figure Legend Snippet: PAI1 inhibits binding of EA-tPA to immunopurified NMDA-R. (A) Extracts of mouse cortex were incubated with NMDA-R NR1 subunit-specific antibody (NR1) or with NS-IgG (IgG) coupled to Protein-G–Dynabeads. Bound proteins were immunoprecipitated in

    Techniques Used: Binding Assay, Incubation, Immunoprecipitation

    7) Product Images from "Glia maturation factor-γ regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS"

    Article Title: Glia maturation factor-γ regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2018026070

    GMFG is associated with the mitochondrial membrane protein ATAD3A. (A) Cellular lysates of human THP-1 cells transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours were immunoprecipitated with control immunoglobulin G (IgG) or monoclonal anti-GMFG antibodies. The immunoprecipitated proteins were isolated using Dynabeads protein G, eluted, and subjected to liquid chromatography-tandem mass spectrometry analysis. Proteome data from Ctrl siRNA- or GMFG siRNA-transfected THP-1 cells were analyzed by MaxQuant, including protein name, intensity L (Ctrl siRNA- or GMFG siRNA-transfected cells immunoprecipitated with IgG antibody), intensity H (Ctrl siRNA-transfected cells immunoprecipitated with GMFG antibody), intensity M (GMFG siRNA-transfected cells immunoprecipitated with GMFG antibody), normalized H/L ratio, H/M ratio, and M/L ratio. (B-C) Immunoprecipitation analysis of GMFG association with the mitochondrial membrane protein ATAD3A in human THP-1 cells. Total cellular lysates were immunoprecipitated with anti-GMFG antibody (B) or anti-ATAD3A antibody (C), then the immunoprecipitants subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates) are shown in the left 2 lanes; immunoprecipitates (IPs) are shown in the right 2 lanes. (D) Cellular lysates of human HEK-293T cells cotransfected with GFP-tagged GMFG plasmid and Myc-DDK–tagged ATAD3A plasmid for 48 hours were immunoprecipitated with control IgG, anti-GFP, or anti-Myc antibody; the IPs were then subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates; bottom). Each sample corresponds to 5% of the cell lysate used in each immunoprecipitation. (E) Immunoblot analysis of ATAD3A proteins in cellular lysates of RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 (LPS/IFN-γ) or M2 (IL-4/IL-13) macrophage inducers for 24 hours. α-tubulin was used as a loading control.
    Figure Legend Snippet: GMFG is associated with the mitochondrial membrane protein ATAD3A. (A) Cellular lysates of human THP-1 cells transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours were immunoprecipitated with control immunoglobulin G (IgG) or monoclonal anti-GMFG antibodies. The immunoprecipitated proteins were isolated using Dynabeads protein G, eluted, and subjected to liquid chromatography-tandem mass spectrometry analysis. Proteome data from Ctrl siRNA- or GMFG siRNA-transfected THP-1 cells were analyzed by MaxQuant, including protein name, intensity L (Ctrl siRNA- or GMFG siRNA-transfected cells immunoprecipitated with IgG antibody), intensity H (Ctrl siRNA-transfected cells immunoprecipitated with GMFG antibody), intensity M (GMFG siRNA-transfected cells immunoprecipitated with GMFG antibody), normalized H/L ratio, H/M ratio, and M/L ratio. (B-C) Immunoprecipitation analysis of GMFG association with the mitochondrial membrane protein ATAD3A in human THP-1 cells. Total cellular lysates were immunoprecipitated with anti-GMFG antibody (B) or anti-ATAD3A antibody (C), then the immunoprecipitants subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates) are shown in the left 2 lanes; immunoprecipitates (IPs) are shown in the right 2 lanes. (D) Cellular lysates of human HEK-293T cells cotransfected with GFP-tagged GMFG plasmid and Myc-DDK–tagged ATAD3A plasmid for 48 hours were immunoprecipitated with control IgG, anti-GFP, or anti-Myc antibody; the IPs were then subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates; bottom). Each sample corresponds to 5% of the cell lysate used in each immunoprecipitation. (E) Immunoblot analysis of ATAD3A proteins in cellular lysates of RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 (LPS/IFN-γ) or M2 (IL-4/IL-13) macrophage inducers for 24 hours. α-tubulin was used as a loading control.

    Techniques Used: Transfection, Immunoprecipitation, Isolation, Liquid Chromatography, Mass Spectrometry, Plasmid Preparation

    8) Product Images from "The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus"

    Article Title: The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03347

    YpkA and SakA interact in vivo during heat shock stress. The wild-type and sakA ::GFP ypkA ::3xHA strains were used in the Co-IP assays. Strains were grown at 30°C (24 h) and subsequently exposed to heat shock at 48°C for the indicated times. (A) GFP-Trap resin was used to immunoprecipitate SakA::GFP. (B) Dynabeads Protein A were incubated with monoclonal α-HA antibody and used to immunoprecipitate YpkA::3xHA in reciprocal experiment. Co-immunoprecipitated proteins were investigated via Western blot analysis using α-HA and α-GFP antibodies. The Coomassie Brilliant Blue (CBB) stained gel was used as an additional loading sample control.
    Figure Legend Snippet: YpkA and SakA interact in vivo during heat shock stress. The wild-type and sakA ::GFP ypkA ::3xHA strains were used in the Co-IP assays. Strains were grown at 30°C (24 h) and subsequently exposed to heat shock at 48°C for the indicated times. (A) GFP-Trap resin was used to immunoprecipitate SakA::GFP. (B) Dynabeads Protein A were incubated with monoclonal α-HA antibody and used to immunoprecipitate YpkA::3xHA in reciprocal experiment. Co-immunoprecipitated proteins were investigated via Western blot analysis using α-HA and α-GFP antibodies. The Coomassie Brilliant Blue (CBB) stained gel was used as an additional loading sample control.

    Techniques Used: In Vivo, Co-Immunoprecipitation Assay, Incubation, Immunoprecipitation, Western Blot, Staining

    9) Product Images from "Two E3 ligases antagonistically regulate the UV-B response in Arabidopsis"

    Article Title: Two E3 ligases antagonistically regulate the UV-B response in Arabidopsis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1816268116

    COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.
    Figure Legend Snippet: COP1 degrades RUP1 and RUP2 under UV-B light. ( A ) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( B ) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. ( C ) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. ( D and E ) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/ cop1-4 ( D ), or FLAG-RUP1 YFP-COP1 ( E ) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.

    Techniques Used: Negative Control, In Vitro, Purification, Incubation, Transfection, Co-Immunoprecipitation Assay, In Vivo, Magnetic Beads

    10) Product Images from "HIV Protease Inhibitors Alter Amyloid Precursor Protein Processing via β-Site Amyloid Precursor Protein Cleaving Enzyme-1 Translational Up-Regulation"

    Article Title: HIV Protease Inhibitors Alter Amyloid Precursor Protein Processing via β-Site Amyloid Precursor Protein Cleaving Enzyme-1 Translational Up-Regulation

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2016.09.006

    Ritonavir does not affect BACE1 protein stability in primary neurons. A: Primary rat neurons were pulse labeled for 30 minutes with 35 S-methionine/cysteine-containing Dulbecco's modified Eagle's medium and chased with neurobasal media plus B27. Radiolabeled BACE1 was immunoprecipitated with Dynabeads Protein A and 2 μg anti-BACE1 12 hours after labeling to confirm enrichment. B: Neurons were pulse labeled, as in A , and chased with neurobasal media with or without 10 μmol/L ritonavir for 24 hours. BACE1 was immunoprecipitated, as in A Representative autoradiogram shown. C: The levels of 35 S-methionine/cysteine-labeled BACE1 were quantified and the percentage changes, relative to 0 hour time point, are plotted. No significant differences in BACE1 half-life were observed between control and ritonavir-treated neurons ( P > 0.05, linear regression analysis). All values represent means ± SEM ( C ). n = 3 ( B and C ). M, molecular weight; mAb, monoclonal antibody; ms, mouse; rb, rabbit; WC, whole cell.
    Figure Legend Snippet: Ritonavir does not affect BACE1 protein stability in primary neurons. A: Primary rat neurons were pulse labeled for 30 minutes with 35 S-methionine/cysteine-containing Dulbecco's modified Eagle's medium and chased with neurobasal media plus B27. Radiolabeled BACE1 was immunoprecipitated with Dynabeads Protein A and 2 μg anti-BACE1 12 hours after labeling to confirm enrichment. B: Neurons were pulse labeled, as in A , and chased with neurobasal media with or without 10 μmol/L ritonavir for 24 hours. BACE1 was immunoprecipitated, as in A Representative autoradiogram shown. C: The levels of 35 S-methionine/cysteine-labeled BACE1 were quantified and the percentage changes, relative to 0 hour time point, are plotted. No significant differences in BACE1 half-life were observed between control and ritonavir-treated neurons ( P > 0.05, linear regression analysis). All values represent means ± SEM ( C ). n = 3 ( B and C ). M, molecular weight; mAb, monoclonal antibody; ms, mouse; rb, rabbit; WC, whole cell.

    Techniques Used: Labeling, Modification, Immunoprecipitation, Molecular Weight, Mass Spectrometry

    11) Product Images from "Glia maturation factor-γ regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS"

    Article Title: Glia maturation factor-γ regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2018026070

    GMFG is associated with the mitochondrial membrane protein ATAD3A. (A) Cellular lysates of human THP-1 cells transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours were immunoprecipitated with control immunoglobulin G (IgG) or monoclonal anti-GMFG antibodies. The immunoprecipitated proteins were isolated using Dynabeads protein G, eluted, and subjected to liquid chromatography-tandem mass spectrometry analysis. Proteome data from Ctrl siRNA- or GMFG siRNA-transfected THP-1 cells were analyzed by MaxQuant, including protein name, intensity L (Ctrl siRNA- or GMFG siRNA-transfected cells immunoprecipitated with IgG antibody), intensity H (Ctrl siRNA-transfected cells immunoprecipitated with GMFG antibody), intensity M (GMFG siRNA-transfected cells immunoprecipitated with GMFG antibody), normalized H/L ratio, H/M ratio, and M/L ratio. (B-C) Immunoprecipitation analysis of GMFG association with the mitochondrial membrane protein ATAD3A in human THP-1 cells. Total cellular lysates were immunoprecipitated with anti-GMFG antibody (B) or anti-ATAD3A antibody (C), then the immunoprecipitants subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates) are shown in the left 2 lanes; immunoprecipitates (IPs) are shown in the right 2 lanes. (D) Cellular lysates of human HEK-293T cells cotransfected with GFP-tagged GMFG plasmid and Myc-DDK–tagged ATAD3A plasmid for 48 hours were immunoprecipitated with control IgG, anti-GFP, or anti-Myc antibody; the IPs were then subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates; bottom). Each sample corresponds to 5% of the cell lysate used in each immunoprecipitation. (E) Immunoblot analysis of ATAD3A proteins in cellular lysates of RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 (LPS/IFN-γ) or M2 (IL-4/IL-13) macrophage inducers for 24 hours. α-tubulin was used as a loading control.
    Figure Legend Snippet: GMFG is associated with the mitochondrial membrane protein ATAD3A. (A) Cellular lysates of human THP-1 cells transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours were immunoprecipitated with control immunoglobulin G (IgG) or monoclonal anti-GMFG antibodies. The immunoprecipitated proteins were isolated using Dynabeads protein G, eluted, and subjected to liquid chromatography-tandem mass spectrometry analysis. Proteome data from Ctrl siRNA- or GMFG siRNA-transfected THP-1 cells were analyzed by MaxQuant, including protein name, intensity L (Ctrl siRNA- or GMFG siRNA-transfected cells immunoprecipitated with IgG antibody), intensity H (Ctrl siRNA-transfected cells immunoprecipitated with GMFG antibody), intensity M (GMFG siRNA-transfected cells immunoprecipitated with GMFG antibody), normalized H/L ratio, H/M ratio, and M/L ratio. (B-C) Immunoprecipitation analysis of GMFG association with the mitochondrial membrane protein ATAD3A in human THP-1 cells. Total cellular lysates were immunoprecipitated with anti-GMFG antibody (B) or anti-ATAD3A antibody (C), then the immunoprecipitants subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates) are shown in the left 2 lanes; immunoprecipitates (IPs) are shown in the right 2 lanes. (D) Cellular lysates of human HEK-293T cells cotransfected with GFP-tagged GMFG plasmid and Myc-DDK–tagged ATAD3A plasmid for 48 hours were immunoprecipitated with control IgG, anti-GFP, or anti-Myc antibody; the IPs were then subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates; bottom). Each sample corresponds to 5% of the cell lysate used in each immunoprecipitation. (E) Immunoblot analysis of ATAD3A proteins in cellular lysates of RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 (LPS/IFN-γ) or M2 (IL-4/IL-13) macrophage inducers for 24 hours. α-tubulin was used as a loading control.

    Techniques Used: Transfection, Immunoprecipitation, Isolation, Liquid Chromatography, Mass Spectrometry, Plasmid Preparation

    12) Product Images from "Protein Phosphatase 1α Interacts with Venezuelan Equine Encephalitis Virus Capsid Protein and Regulates Viral Replication through Modulation of Capsid Phosphorylation"

    Article Title: Protein Phosphatase 1α Interacts with Venezuelan Equine Encephalitis Virus Capsid Protein and Regulates Viral Replication through Modulation of Capsid Phosphorylation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02068-17

    VEEV capsid is phosphorylated, and this phosphorylation is modulated by PP1. (A) Vero cells were infected with VEEV TC-83 (MOI of 1.0) for 24 h and treated with DMSO or 1E7-03 (10 μM). Cells were lysed and immunoprecipitated with anti-HA or anti-VEEV capsid antibodies. Protein complexes were bound to protein G Dynabeads. Samples were run on SDS-PAGE and Western blotted for phospho-Ser or phospho-Thr residues. (B) Quantitation of Western blots. Western blot band density was analyzed on Bio-Rad Quantity One software and normalized to that of capsid. Normalized values were calculated relative to those for DMSO-treated cells. Phosphoserine phosphorylation increased by ∼27% after 1E7-03 treatment and phosphothreonine increased by ∼31% after treatment with 1E7-03. *, P
    Figure Legend Snippet: VEEV capsid is phosphorylated, and this phosphorylation is modulated by PP1. (A) Vero cells were infected with VEEV TC-83 (MOI of 1.0) for 24 h and treated with DMSO or 1E7-03 (10 μM). Cells were lysed and immunoprecipitated with anti-HA or anti-VEEV capsid antibodies. Protein complexes were bound to protein G Dynabeads. Samples were run on SDS-PAGE and Western blotted for phospho-Ser or phospho-Thr residues. (B) Quantitation of Western blots. Western blot band density was analyzed on Bio-Rad Quantity One software and normalized to that of capsid. Normalized values were calculated relative to those for DMSO-treated cells. Phosphoserine phosphorylation increased by ∼27% after 1E7-03 treatment and phosphothreonine increased by ∼31% after treatment with 1E7-03. *, P

    Techniques Used: Infection, Immunoprecipitation, SDS Page, Western Blot, Quantitation Assay, Software

    PP1 interacts with VEEV capsid. (A) Vero cells were infected with VEEV TC-83 (MOI, 1.0) for 24 h. Cells were lysed and 1 μg of either anti-HA or anti-PP1α was added to 1 mg of protein. Protein complexes were bound to protein G Dynabeads, Samples were run on SDS-PAGE and Western blotted for capsid or PP1α. (B) Vero cells were infected with VEEV TC-83 (MOI, 1.0) or transfected with 30 μg of V5-tagged capsid plasmid. Cells were collected 24 h after infection or transfection and lysed, and 1 μg of either anti-HA or anti-capsid was added to 1 mg of protein. Protein complexes were bound to protein G Dynabeads, and samples were run on SDS-PAGE and Western blotted for PP1α or capsid. Capsid input was imaged separately due to high levels of capsid in the input samples. (C) Vero cells were transfected with the VEEV structural polyprotein plasmid and treated with 10 μM 1E7-03 or DMSO. Cells were collected 24 h after transfection and lysed, and 1 μg of anti-HA or anti-PP1α was added to 1 mg of protein. Protein complexes were bound to protein G Dynabeads. Samples were run on SDS-PAGE and Western blotted for PP1α or capsid. Images are representative of those from 3 biological replicates.
    Figure Legend Snippet: PP1 interacts with VEEV capsid. (A) Vero cells were infected with VEEV TC-83 (MOI, 1.0) for 24 h. Cells were lysed and 1 μg of either anti-HA or anti-PP1α was added to 1 mg of protein. Protein complexes were bound to protein G Dynabeads, Samples were run on SDS-PAGE and Western blotted for capsid or PP1α. (B) Vero cells were infected with VEEV TC-83 (MOI, 1.0) or transfected with 30 μg of V5-tagged capsid plasmid. Cells were collected 24 h after infection or transfection and lysed, and 1 μg of either anti-HA or anti-capsid was added to 1 mg of protein. Protein complexes were bound to protein G Dynabeads, and samples were run on SDS-PAGE and Western blotted for PP1α or capsid. Capsid input was imaged separately due to high levels of capsid in the input samples. (C) Vero cells were transfected with the VEEV structural polyprotein plasmid and treated with 10 μM 1E7-03 or DMSO. Cells were collected 24 h after transfection and lysed, and 1 μg of anti-HA or anti-PP1α was added to 1 mg of protein. Protein complexes were bound to protein G Dynabeads. Samples were run on SDS-PAGE and Western blotted for PP1α or capsid. Images are representative of those from 3 biological replicates.

    Techniques Used: Infection, SDS Page, Western Blot, Transfection, Plasmid Preparation

    13) Product Images from "The lipid peroxidation product 4-hydroxynonenal contributes to oxidative stress-mediated deterioration of the ageing oocyte"

    Article Title: The lipid peroxidation product 4-hydroxynonenal contributes to oxidative stress-mediated deterioration of the ageing oocyte

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-06372-z

    Examination of α-, β- and γ-tubulin/4-HNE interaction in GV and MII oocytes. Co-localisation, immunoprecipitation and PLA experiments were employed to examine the interaction between tubulins and 4-HNE. ( a ) For co-localisation, GV oocytes were untreated or treated with 35 μM H 2 O 2 for 1 h and then underwent IVM in preparation for fixation at MII. α-tubulin (green) co-localised with 4-HNE (red) at the MII spindle before and after treatment with 35 μM H 2 O 2 . Notably, 4-HNE aggregates also co-localised microtubule asters (white arrows). Scale bar = 20 μm. ( b ) γ-tubulin (green) also co-localised with 4-HNE (red) at the poles of the MII spindle before and after treatment with 35 μM H 2 O 2 . ( c–e ) For immunoprecipitation assays, lysates from H 2 O 2 -treated GV and IVM MII oocytes were incubated with protein G Dynabeads conjugated with anti-tubulin antibodies. The Dynabeads were then washed and proteins that bound were eluted for immunoblotting on two mirrored membranes. One membrane (panel 1) was probed with anti-tubulin antibodies confirming the effectiveness of the immunoprecipitation. The alternate membrane was probed with 4-HNE antibodies (panel 2). Whole oocyte lysate was used as a positive control. Antibody-only control (ab only) and preclear elute negative controls were used to confirm specificity of elution. ( f ) PLA between 4-HNE and α-, β- or γ-tubulin revealed fluorescent foci at the MII spindle indicating an intimate association between 4-HNE and tubulins. Scale bar = 20 μm. Nuclei were counterstained with Hoechst (blue). Experiments were performed with three biological replicates with each replicate comprising between 10–30 oocytes pooled from a minimum of three animals. Immunoprecipitation experiments was performed in technical duplicate from between 350–500 oocytes pooled from between 8–12 mice.
    Figure Legend Snippet: Examination of α-, β- and γ-tubulin/4-HNE interaction in GV and MII oocytes. Co-localisation, immunoprecipitation and PLA experiments were employed to examine the interaction between tubulins and 4-HNE. ( a ) For co-localisation, GV oocytes were untreated or treated with 35 μM H 2 O 2 for 1 h and then underwent IVM in preparation for fixation at MII. α-tubulin (green) co-localised with 4-HNE (red) at the MII spindle before and after treatment with 35 μM H 2 O 2 . Notably, 4-HNE aggregates also co-localised microtubule asters (white arrows). Scale bar = 20 μm. ( b ) γ-tubulin (green) also co-localised with 4-HNE (red) at the poles of the MII spindle before and after treatment with 35 μM H 2 O 2 . ( c–e ) For immunoprecipitation assays, lysates from H 2 O 2 -treated GV and IVM MII oocytes were incubated with protein G Dynabeads conjugated with anti-tubulin antibodies. The Dynabeads were then washed and proteins that bound were eluted for immunoblotting on two mirrored membranes. One membrane (panel 1) was probed with anti-tubulin antibodies confirming the effectiveness of the immunoprecipitation. The alternate membrane was probed with 4-HNE antibodies (panel 2). Whole oocyte lysate was used as a positive control. Antibody-only control (ab only) and preclear elute negative controls were used to confirm specificity of elution. ( f ) PLA between 4-HNE and α-, β- or γ-tubulin revealed fluorescent foci at the MII spindle indicating an intimate association between 4-HNE and tubulins. Scale bar = 20 μm. Nuclei were counterstained with Hoechst (blue). Experiments were performed with three biological replicates with each replicate comprising between 10–30 oocytes pooled from a minimum of three animals. Immunoprecipitation experiments was performed in technical duplicate from between 350–500 oocytes pooled from between 8–12 mice.

    Techniques Used: Immunoprecipitation, Proximity Ligation Assay, Incubation, Positive Control, Mouse Assay

    Related Articles

    Transduction:

    Article Title: Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity
    Article Snippet: Materials Heparinase III (H8891), chondroitinase ABC (C2905), 4-Nitrophenyl β-D-xylopyranoside (PNP-Xyl; N2132), CS-4 sodium salt from bovine trachea (C9819), heparin, biotin-HRP (S3438), heparin-agarose (H0402), G-418 disulfate salt (A 8601), puromycin (P9620), Ca9 shRNA MISSION lentiviral transduction particle (SHCLNV; TRCN0000150123), MISSION pLKO.1-puro Non-Mammalian shRNA Control Transduction Particles (SHC002V), iodoacetamide, and FITC-conjugated 10 kDa dextran were from Sigma-Aldrich. .. Alexa Fluor (AF)-conjugated secondary IgG antibodies, Transferrin-AF488 (T13342), streptavidin-AF-488 (S32354), immunoprecipitation Kit-Dynabeads® Protein G (10007D), Cholera toxin-subunit B-AF-488 (C34775), biotin EZ-Link Sulfo-NHS-SS-Biotin (21331), streptavidin Sepharose High Performance (17-5113-01), NuPage 4-12% Bis Tris gels, SeeBlue Plus2, and MesNa (sodium-2-mercaptoethanesulfonate) were from Thermo Fisher Scientific.

    Centrifugation:

    Article Title: Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer
    Article Snippet: The cells were lysed in a buffer containing 50 mM Tris (pH 7.5), 250 mM NaCl, 0.1% Triton X (Sigma-Aldrich Corporation), 1 mM ethylenediaminetetraacetic acid, 50 mM NaF, and protease and phosphatase inhibitor cocktails (Thermo Scientific™ Haltliquid), and centrifuged at 12,000 g for 20 min. Supernatants were immunoprecipitated using the Dynabeads Protein G immunoprecipitation kit (Invitrogen Corporation), according to the manufacturer’s instructions, with N-cadherin antibody (610920, BD Transduction Laboratories), and DcR-2 antibody (D13H4, Cell Signaling Technology, Inc.). .. Beads were collected by brief centrifugation, washed four times with buffer, boiled in sodium dodecyl sulfate (SDS) gel loading buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane (Schleicher & Schuell BioScience GmbH, Dasse, Germany) by electroblotting.

    Article Title: Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins
    Article Snippet: Cells were harvested 3 dpi using a rubber policeman and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA and 1% NP-40) at 4 °C for 30 min, followed by centrifugation at 1150× g for 5 min. Post-nuclear supernatants were immunoprecipitated overnight with polyclonal antibodies against ISAV s8ORF2 [ ] or matrix (M) [ ], ISAV fusion protein (F) [ ] or monoclonal antibody against NP (anti-ISAV, Aquatic Diagnostics). .. The following day pull-down was performed using the Dynabeads Protein G immunoprecipitation kit (Novex, Thermo-Fisher, Waltham, MA, USA ) following the protocol by the manufacturer.

    Article Title: A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation
    Article Snippet: Three days after infection, whole cells were resuspended in lysis buffer and clarified by centrifugation (5 min, 2,000 × g ). .. Immunoprecipitation was performed at room temperature with anti-FLAG (Sigma) or anti-V5 (Abcam) antibodies using a Dynabeads protein G immunoprecipitation kit (Thermo Fisher).

    Amplification:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol. .. A total of 200 ng of IN and IP DNA was amplified with the Affymetrix Chromatin Immunoprecipitation Assay Protocol.

    SDS-Gel:

    Article Title: Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer
    Article Snippet: The cells were lysed in a buffer containing 50 mM Tris (pH 7.5), 250 mM NaCl, 0.1% Triton X (Sigma-Aldrich Corporation), 1 mM ethylenediaminetetraacetic acid, 50 mM NaF, and protease and phosphatase inhibitor cocktails (Thermo Scientific™ Haltliquid), and centrifuged at 12,000 g for 20 min. Supernatants were immunoprecipitated using the Dynabeads Protein G immunoprecipitation kit (Invitrogen Corporation), according to the manufacturer’s instructions, with N-cadherin antibody (610920, BD Transduction Laboratories), and DcR-2 antibody (D13H4, Cell Signaling Technology, Inc.). .. Beads were collected by brief centrifugation, washed four times with buffer, boiled in sodium dodecyl sulfate (SDS) gel loading buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane (Schleicher & Schuell BioScience GmbH, Dasse, Germany) by electroblotting.

    Incubation:

    Article Title: Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia 1-Induced Proinflammatory Signaling in Microglia 1 2-Induced Proinflammatory Signaling in Microglia 1 2 3
    Article Snippet: Co-immunoprecipitation or pull down experiments were performed using DynaBeads Protein G [35 μl of Dynabeads/sample (100–400 μg protein), Life Technologies, 10007D], and a magnetic rack (Life Technologies). .. The beads were washed in RIPA buffer and incubated with 5 μg primary antibody (in 200 μl PBS for 30 min at room temperature).

    Article Title: Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer
    Article Snippet: The cells were lysed in a buffer containing 50 mM Tris (pH 7.5), 250 mM NaCl, 0.1% Triton X (Sigma-Aldrich Corporation), 1 mM ethylenediaminetetraacetic acid, 50 mM NaF, and protease and phosphatase inhibitor cocktails (Thermo Scientific™ Haltliquid), and centrifuged at 12,000 g for 20 min. Supernatants were immunoprecipitated using the Dynabeads Protein G immunoprecipitation kit (Invitrogen Corporation), according to the manufacturer’s instructions, with N-cadherin antibody (610920, BD Transduction Laboratories), and DcR-2 antibody (D13H4, Cell Signaling Technology, Inc.). .. The primary antibodies were diluted in PBS containing 5% non-fat dry milk powder and incubated overnight at 4° C. Then, the strips were washed and incubated with the secondary antibody for 30 min at room temperature.

    Article Title: Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65
    Article Snippet: Coimmunoprecipitation assay was performed using Dynabeads protein G Immunoprecipitation Kit (Invitrogen). .. The supernates were incubated with anti-FLAG Ab, anti-p65 Ab, mouse IgG Ab, or rabbit IgG Ab overnight at 4 °C, as indicated.

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation
    Article Snippet: Co-immunoprecipitation or pull-down experiments were carried out using DynaBeads Protein G [35 μl of Dynabeads®/sample (100–250 μg protein), Life Technologies, 10007D] and a magnetic rack (Life Technologies). .. Briefly, the beads were washed in RIPA buffer and incubated with 5 μg primary antibody (in 200 μl PBS for 1 h rotating at room temperature) and washed 3 times for 5 min in PBS buffer before equal amounts of protein (100–250 μg) were incubated for 2 h at room temperature (in a total volume of 750 μl).

    Proliferation Assay:

    Article Title: Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity
    Article Snippet: Alexa Fluor (AF)-conjugated secondary IgG antibodies, Transferrin-AF488 (T13342), streptavidin-AF-488 (S32354), immunoprecipitation Kit-Dynabeads® Protein G (10007D), Cholera toxin-subunit B-AF-488 (C34775), biotin EZ-Link Sulfo-NHS-SS-Biotin (21331), streptavidin Sepharose High Performance (17-5113-01), NuPage 4-12% Bis Tris gels, SeeBlue Plus2, and MesNa (sodium-2-mercaptoethanesulfonate) were from Thermo Fisher Scientific. .. QuickChange II Site-directed mutagenesis Kit (200523) was from Agilent Technologies, and CellTiter 96® AQueous One Solution Cell Proliferation Assay MTS reagent from Promega.

    Mass Spectrometry:

    Article Title: A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation
    Article Snippet: Paragraph title: Immunoprecipitation, Co-immunoprecipitation, and Mass Spectrometry ... Immunoprecipitation was performed at room temperature with anti-FLAG (Sigma) or anti-V5 (Abcam) antibodies using a Dynabeads protein G immunoprecipitation kit (Thermo Fisher).

    Modification:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: MeDip-chip The global DNA methylation profiles of cells treated with sorafenib or left untreated were obtained by methylated DNA immunoprecipitation coupled with Affymetrix Human Promoter 1.0R Tiling Arrays (MeDip-chip) using a modification of the Affymetrix chromatin immunoprecipitation assay protocol. .. The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol.

    Western Blot:

    Article Title: Survivin Monomer Plays an Essential Role in Apoptosis Regulation *
    Article Snippet: Paragraph title: Immunoprecipitation and Western Blot Analysis ... Cells were lysed as described previously , and immunoprecipitation was performed using a Dynabeads-protein G immunoprecipitation kit (Invitrogen) as instructed by the manufacturer.

    Article Title: Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer
    Article Snippet: Paragraph title: Western blot and immunoprecipitation analysis ... The cells were lysed in a buffer containing 50 mM Tris (pH 7.5), 250 mM NaCl, 0.1% Triton X (Sigma-Aldrich Corporation), 1 mM ethylenediaminetetraacetic acid, 50 mM NaF, and protease and phosphatase inhibitor cocktails (Thermo Scientific™ Haltliquid), and centrifuged at 12,000 g for 20 min. Supernatants were immunoprecipitated using the Dynabeads Protein G immunoprecipitation kit (Invitrogen Corporation), according to the manufacturer’s instructions, with N-cadherin antibody (610920, BD Transduction Laboratories), and DcR-2 antibody (D13H4, Cell Signaling Technology, Inc.).

    Article Title: Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms
    Article Snippet: Immunoprecipitations were performed using the Dynabeads® Protein G immunoprecipitation kit (Invitrogen) according to the manufacturer's instructions using an anti-α1 sGC (GC1) antibody (Sigma-Aldrich). .. Sample lysates were then analyzed by Western blot using an anti-nNOS antibody (BD Biosciences).

    Article Title: Epidermal development, growth control, and homeostasis in the face of centrosome amplification
    Article Snippet: Paragraph title: Western Blot and Immunoprecipitation Analyses. ... Immunoprecipitations were performed using Dynabeads Protein G Immunoprecipitation Kit (Novex/Life Technologies) according to the manufacturer’s protocol, and samples were loaded for equal volume.

    Over Expression:

    Article Title: Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia 1-Induced Proinflammatory Signaling in Microglia 1 2-Induced Proinflammatory Signaling in Microglia 1 2 3
    Article Snippet: Co-immunoprecipitation and pull-down experiments Overexpression of tumor necrosis factor receptor associated factor 6 (TRAF6) was performed by transfection of the pCMV5-FLAG-wt-TRAF6 vector in BV-2 cells. .. Co-immunoprecipitation or pull down experiments were performed using DynaBeads Protein G [35 μl of Dynabeads/sample (100–400 μg protein), Life Technologies, 10007D], and a magnetic rack (Life Technologies).

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation
    Article Snippet: Co-immunoprecipitation Overexpression of TRAF6-FLAG, Smad6-FLAG, or Ubiquitin-HA was carried out by transfection of 2.5 μg plasmid/9 × 105 cells in a T75 flask, using lipofectamine. .. Co-immunoprecipitation or pull-down experiments were carried out using DynaBeads Protein G [35 μl of Dynabeads®/sample (100–250 μg protein), Life Technologies, 10007D] and a magnetic rack (Life Technologies).

    Hybridization:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol. .. Hybridization on Human Promoter 1.0R array was performed using the reagents contained in GeneChip® Hybridization, Wash, and Stain kit (Affymetrix) using GeneChip hybridization oven 640 (Affymetrix).

    Transfection:

    Article Title: Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia 1-Induced Proinflammatory Signaling in Microglia 1 2-Induced Proinflammatory Signaling in Microglia 1 2 3
    Article Snippet: In short, BV-2 cells were transfected with FLAG-wt-TRAF6 (Addgene, 21624) or pCMV5-FLAG (1.5 µg/3 × 105 cells) using Lipofectamine. .. Co-immunoprecipitation or pull down experiments were performed using DynaBeads Protein G [35 μl of Dynabeads/sample (100–400 μg protein), Life Technologies, 10007D], and a magnetic rack (Life Technologies).

    Article Title: Autophagy-Associated Proteins Control Ebola Virus Internalization Into Host Cells
    Article Snippet: Immunoprecipitation siRNA-transfected HeLa cells grown in 100-mm dishes were transfected with 3 μg of pCMV-myc-LC3 and 3 μg of either pEYFP-Ankfy1 or pEYFP-C1. .. Proteins were precipitated using a rabbit anti-eGFP antibody and Dynabeads protein G immunoprecipitation kit (Thermo Fisher) according to the manufacturer’s protocol.

    Article Title: Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65
    Article Snippet: For coimmunoprecipitation experiments, 293T cells were transfected with indicated plasmids. .. Coimmunoprecipitation assay was performed using Dynabeads protein G Immunoprecipitation Kit (Invitrogen).

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation
    Article Snippet: Twenty hours post transfection, the cells were stimulated with Pam3 CSK4 (100 ng/ml), PolyI:C (100 ng/ml), or LPS (100 ng/ml) for 1 h before lysis in RIPA buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma). .. Co-immunoprecipitation or pull-down experiments were carried out using DynaBeads Protein G [35 μl of Dynabeads®/sample (100–250 μg protein), Life Technologies, 10007D] and a magnetic rack (Life Technologies).

    Immunoprecipitation:

    Article Title: Survivin Monomer Plays an Essential Role in Apoptosis Regulation *
    Article Snippet: .. Cells were lysed as described previously , and immunoprecipitation was performed using a Dynabeads-protein G immunoprecipitation kit (Invitrogen) as instructed by the manufacturer. ..

    Article Title: Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer
    Article Snippet: .. The cells were lysed in a buffer containing 50 mM Tris (pH 7.5), 250 mM NaCl, 0.1% Triton X (Sigma-Aldrich Corporation), 1 mM ethylenediaminetetraacetic acid, 50 mM NaF, and protease and phosphatase inhibitor cocktails (Thermo Scientific™ Haltliquid), and centrifuged at 12,000 g for 20 min. Supernatants were immunoprecipitated using the Dynabeads Protein G immunoprecipitation kit (Invitrogen Corporation), according to the manufacturer’s instructions, with N-cadherin antibody (610920, BD Transduction Laboratories), and DcR-2 antibody (D13H4, Cell Signaling Technology, Inc.). .. Beads were collected by brief centrifugation, washed four times with buffer, boiled in sodium dodecyl sulfate (SDS) gel loading buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane (Schleicher & Schuell BioScience GmbH, Dasse, Germany) by electroblotting.

    Article Title: Astrocyte-derived pentraxin 3 supports blood-brain barrier integrity under acute phase of stroke
    Article Snippet: .. According to the manufacture’s instruction, anti-rabbit PTX3 antibody (Enzo) and Dynabeads Protein G Immunoprecipitation kit (Life technologies) were used to precipitate PTX3 in culture media. .. RBE.4 monolayer was prepared on collagen-I-coated transwells (6.5mm diameter, 3.0µm pore size polycarbonate filter, Corning).

    Article Title: Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins
    Article Snippet: .. The following day pull-down was performed using the Dynabeads Protein G immunoprecipitation kit (Novex, Thermo-Fisher, Waltham, MA, USA ) following the protocol by the manufacturer. .. For SDS-PAGE, magnetic bead pellets were boiled for 10 min in NuPAGE loading buffer with reducing agent (BioRad, Hercules, CA, USA).

    Article Title: Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms
    Article Snippet: .. Immunoprecipitations were performed using the Dynabeads® Protein G immunoprecipitation kit (Invitrogen) according to the manufacturer's instructions using an anti-α1 sGC (GC1) antibody (Sigma-Aldrich). ..

    Article Title: Autophagy-Associated Proteins Control Ebola Virus Internalization Into Host Cells
    Article Snippet: .. Proteins were precipitated using a rabbit anti-eGFP antibody and Dynabeads protein G immunoprecipitation kit (Thermo Fisher) according to the manufacturer’s protocol. .. Eluted proteins were resuspended in sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer, boiled, resolved on a gel, and subjected to immunoblotting analysis with mouse anti–myc tag and anti-eGFP antibodies.

    Article Title: Epidermal development, growth control, and homeostasis in the face of centrosome amplification
    Article Snippet: .. Immunoprecipitations were performed using Dynabeads Protein G Immunoprecipitation Kit (Novex/Life Technologies) according to the manufacturer’s protocol, and samples were loaded for equal volume. .. Antibodies used include rabbit anti-GFP (Invitrogen), mouse anti-GFP (Roche), rabbit anti-RFP (MBL), and mouse anti–β-actin (Sigma-Aldrich).

    Article Title: Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65
    Article Snippet: .. Coimmunoprecipitation assay was performed using Dynabeads protein G Immunoprecipitation Kit (Invitrogen). .. The supernates were incubated with anti-FLAG Ab, anti-p65 Ab, mouse IgG Ab, or rabbit IgG Ab overnight at 4 °C, as indicated.

    Article Title: Loss of the Histone Methyltransferase EZH2 induces Resistance to Multiple Drugs in Acute Myeloid Leukemia
    Article Snippet: .. Immunoprecipitation of EZH2 or Ubiquitin was performed using Dynabeads® Protein G Immunoprecipitation Kit (Life Technologies, Darmstadt, Germany) according to manufacturer`s instructions using following antibodies: rabbit IgG control (Cell Signaling), anti-EZH2 (DC29, Cell Signaling) and anti-Ubiquitin antibody (Abcam, ab7780). .. Co-Immunoprecipitation of the CDK1/EZH2/STIP1/HSP90 interaction was performed using Dynabeads® Co-Immunoprecipitation Kit (Life Technologies, Darmstadt, Germany).

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: .. The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol. .. The MeDip was performed in duplicate for each DNA sample starting with initial fragmentation step.

    Article Title: Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity
    Article Snippet: .. Alexa Fluor (AF)-conjugated secondary IgG antibodies, Transferrin-AF488 (T13342), streptavidin-AF-488 (S32354), immunoprecipitation Kit-Dynabeads® Protein G (10007D), Cholera toxin-subunit B-AF-488 (C34775), biotin EZ-Link Sulfo-NHS-SS-Biotin (21331), streptavidin Sepharose High Performance (17-5113-01), NuPage 4-12% Bis Tris gels, SeeBlue Plus2, and MesNa (sodium-2-mercaptoethanesulfonate) were from Thermo Fisher Scientific. .. Protein G agarose beads (sc-2002) was from Santa Cruz Biotechnology, and Myc-αDDK-tagged (FLAG-tag) ORF clone of Homo sapiens Ca9 (RC204839) and rabbit anti-αDDK antibody (TA100023) were from Origene.

    Article Title: A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation
    Article Snippet: .. Immunoprecipitation was performed at room temperature with anti-FLAG (Sigma) or anti-V5 (Abcam) antibodies using a Dynabeads protein G immunoprecipitation kit (Thermo Fisher). .. Death screening is a new method using active selection and sequencing of dead cells A high-quality catalog of genes essential for human OXPHOS is reported NGRN, WBSCR16, RPUSD3, RPUSD4, TRUB2, and FASTKD2 form a 16S rRNA regulatory module

    Protease Inhibitor:

    Article Title: Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia 1-Induced Proinflammatory Signaling in Microglia 1 2-Induced Proinflammatory Signaling in Microglia 1 2 3
    Article Snippet: Cells were lysed in RIPA buffer (Tris 50 mm , NaCl 150 mm , SDS 0.1%, sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma-Aldrich). .. Co-immunoprecipitation or pull down experiments were performed using DynaBeads Protein G [35 μl of Dynabeads/sample (100–400 μg protein), Life Technologies, 10007D], and a magnetic rack (Life Technologies).

    Article Title: Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms
    Article Snippet: Skeletal muscle tissues were lysed in buffer containing 1% Nonidet P-40, 50 m M Tris-HCL, pH 7.4, 150 m M NaCl, 2 m M EGTA, 2 m M PMSF, and protease inhibitor cocktail (Roche Applied Science). .. Immunoprecipitations were performed using the Dynabeads® Protein G immunoprecipitation kit (Invitrogen) according to the manufacturer's instructions using an anti-α1 sGC (GC1) antibody (Sigma-Aldrich).

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation
    Article Snippet: Twenty hours post transfection, the cells were stimulated with Pam3 CSK4 (100 ng/ml), PolyI:C (100 ng/ml), or LPS (100 ng/ml) for 1 h before lysis in RIPA buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma). .. Co-immunoprecipitation or pull-down experiments were carried out using DynaBeads Protein G [35 μl of Dynabeads®/sample (100–250 μg protein), Life Technologies, 10007D] and a magnetic rack (Life Technologies).

    Infection:

    Article Title: Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins
    Article Snippet: Immunoprecipitation (IP) and SDS-PAGE ASK-cells were seeded to 80%–90% confluence in T-162 flasks and the following day infected with 4 mL of ISAV (1:5 dilution). .. The following day pull-down was performed using the Dynabeads Protein G immunoprecipitation kit (Novex, Thermo-Fisher, Waltham, MA, USA ) following the protocol by the manufacturer.

    Article Title: A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation
    Article Snippet: Three days after infection, whole cells were resuspended in lysis buffer and clarified by centrifugation (5 min, 2,000 × g ). .. Immunoprecipitation was performed at room temperature with anti-FLAG (Sigma) or anti-V5 (Abcam) antibodies using a Dynabeads protein G immunoprecipitation kit (Thermo Fisher).

    Methylated DNA Immunoprecipitation:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: Paragraph title: MeDip-chip ... The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol.

    Nucleic Acid Electrophoresis:

    Article Title: Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer
    Article Snippet: The cells were lysed in a buffer containing 50 mM Tris (pH 7.5), 250 mM NaCl, 0.1% Triton X (Sigma-Aldrich Corporation), 1 mM ethylenediaminetetraacetic acid, 50 mM NaF, and protease and phosphatase inhibitor cocktails (Thermo Scientific™ Haltliquid), and centrifuged at 12,000 g for 20 min. Supernatants were immunoprecipitated using the Dynabeads Protein G immunoprecipitation kit (Invitrogen Corporation), according to the manufacturer’s instructions, with N-cadherin antibody (610920, BD Transduction Laboratories), and DcR-2 antibody (D13H4, Cell Signaling Technology, Inc.). .. Beads were collected by brief centrifugation, washed four times with buffer, boiled in sodium dodecyl sulfate (SDS) gel loading buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane (Schleicher & Schuell BioScience GmbH, Dasse, Germany) by electroblotting.

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation
    Article Snippet: Co-immunoprecipitation or pull-down experiments were carried out using DynaBeads Protein G [35 μl of Dynabeads®/sample (100–250 μg protein), Life Technologies, 10007D] and a magnetic rack (Life Technologies). .. Elution of the samples from the beads using the magnetic rack, where the protein was denatured in RIPA buffer plus 1 × Laemmli buffer by incubating for 10 min at 70 °C, was followed by gel electrophoresis.

    Methylation:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: .. The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol. .. The MeDip was performed in duplicate for each DNA sample starting with initial fragmentation step.

    Mutagenesis:

    Article Title: Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity
    Article Snippet: Alexa Fluor (AF)-conjugated secondary IgG antibodies, Transferrin-AF488 (T13342), streptavidin-AF-488 (S32354), immunoprecipitation Kit-Dynabeads® Protein G (10007D), Cholera toxin-subunit B-AF-488 (C34775), biotin EZ-Link Sulfo-NHS-SS-Biotin (21331), streptavidin Sepharose High Performance (17-5113-01), NuPage 4-12% Bis Tris gels, SeeBlue Plus2, and MesNa (sodium-2-mercaptoethanesulfonate) were from Thermo Fisher Scientific. .. QuickChange II Site-directed mutagenesis Kit (200523) was from Agilent Technologies, and CellTiter 96® AQueous One Solution Cell Proliferation Assay MTS reagent from Promega.

    Isolation:

    Article Title: A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation
    Article Snippet: For protein isolation, the eluate was precipitated using trichloroacetic acid (TCA) and analyzed by mass spectrometry at the Whitehead proteomics facility or by protein immunoblotting. .. Immunoprecipitation was performed at room temperature with anti-FLAG (Sigma) or anti-V5 (Abcam) antibodies using a Dynabeads protein G immunoprecipitation kit (Thermo Fisher).

    Purification:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: .. The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol. .. The MeDip was performed in duplicate for each DNA sample starting with initial fragmentation step.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Autophagy-Associated Proteins Control Ebola Virus Internalization Into Host Cells
    Article Snippet: Proteins were precipitated using a rabbit anti-eGFP antibody and Dynabeads protein G immunoprecipitation kit (Thermo Fisher) according to the manufacturer’s protocol. .. Eluted proteins were resuspended in sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer, boiled, resolved on a gel, and subjected to immunoblotting analysis with mouse anti–myc tag and anti-eGFP antibodies.

    Lysis:

    Article Title: Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65
    Article Snippet: Cells were harvested and lysed with cell lysis buffer for immunoblotting analysis and immunoprecipitation. .. Coimmunoprecipitation assay was performed using Dynabeads protein G Immunoprecipitation Kit (Invitrogen).

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation
    Article Snippet: Twenty hours post transfection, the cells were stimulated with Pam3 CSK4 (100 ng/ml), PolyI:C (100 ng/ml), or LPS (100 ng/ml) for 1 h before lysis in RIPA buffer (Tris 50 mM, NaCl 150 mM, SDS 0.1%, Sodium-deoxycholate 0.5%, Triton X-100 or NP-40 1%, plus 1:100 Protease Inhibitor, Sigma). .. Co-immunoprecipitation or pull-down experiments were carried out using DynaBeads Protein G [35 μl of Dynabeads®/sample (100–250 μg protein), Life Technologies, 10007D] and a magnetic rack (Life Technologies).

    Article Title: A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation
    Article Snippet: Three days after infection, whole cells were resuspended in lysis buffer and clarified by centrifugation (5 min, 2,000 × g ). .. Immunoprecipitation was performed at room temperature with anti-FLAG (Sigma) or anti-V5 (Abcam) antibodies using a Dynabeads protein G immunoprecipitation kit (Thermo Fisher).

    Chromatin Immunoprecipitation:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: MeDip-chip The global DNA methylation profiles of cells treated with sorafenib or left untreated were obtained by methylated DNA immunoprecipitation coupled with Affymetrix Human Promoter 1.0R Tiling Arrays (MeDip-chip) using a modification of the Affymetrix chromatin immunoprecipitation assay protocol. .. The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol.

    SDS Page:

    Article Title: Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins
    Article Snippet: Paragraph title: 2.1.2. Immunoprecipitation (IP) and SDS-PAGE ... The following day pull-down was performed using the Dynabeads Protein G immunoprecipitation kit (Novex, Thermo-Fisher, Waltham, MA, USA ) following the protocol by the manufacturer.

    Plasmid Preparation:

    Article Title: Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia 1-Induced Proinflammatory Signaling in Microglia 1 2-Induced Proinflammatory Signaling in Microglia 1 2 3
    Article Snippet: Co-immunoprecipitation and pull-down experiments Overexpression of tumor necrosis factor receptor associated factor 6 (TRAF6) was performed by transfection of the pCMV5-FLAG-wt-TRAF6 vector in BV-2 cells. .. Co-immunoprecipitation or pull down experiments were performed using DynaBeads Protein G [35 μl of Dynabeads/sample (100–400 μg protein), Life Technologies, 10007D], and a magnetic rack (Life Technologies).

    Article Title: Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation
    Article Snippet: Co-immunoprecipitation Overexpression of TRAF6-FLAG, Smad6-FLAG, or Ubiquitin-HA was carried out by transfection of 2.5 μg plasmid/9 × 105 cells in a T75 flask, using lipofectamine. .. Co-immunoprecipitation or pull-down experiments were carried out using DynaBeads Protein G [35 μl of Dynabeads®/sample (100–250 μg protein), Life Technologies, 10007D] and a magnetic rack (Life Technologies).

    Co-Immunoprecipitation Assay:

    Article Title: Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65
    Article Snippet: .. Coimmunoprecipitation assay was performed using Dynabeads protein G Immunoprecipitation Kit (Invitrogen). .. The supernates were incubated with anti-FLAG Ab, anti-p65 Ab, mouse IgG Ab, or rabbit IgG Ab overnight at 4 °C, as indicated.

    shRNA:

    Article Title: Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity
    Article Snippet: Materials Heparinase III (H8891), chondroitinase ABC (C2905), 4-Nitrophenyl β-D-xylopyranoside (PNP-Xyl; N2132), CS-4 sodium salt from bovine trachea (C9819), heparin, biotin-HRP (S3438), heparin-agarose (H0402), G-418 disulfate salt (A 8601), puromycin (P9620), Ca9 shRNA MISSION lentiviral transduction particle (SHCLNV; TRCN0000150123), MISSION pLKO.1-puro Non-Mammalian shRNA Control Transduction Particles (SHC002V), iodoacetamide, and FITC-conjugated 10 kDa dextran were from Sigma-Aldrich. .. Alexa Fluor (AF)-conjugated secondary IgG antibodies, Transferrin-AF488 (T13342), streptavidin-AF-488 (S32354), immunoprecipitation Kit-Dynabeads® Protein G (10007D), Cholera toxin-subunit B-AF-488 (C34775), biotin EZ-Link Sulfo-NHS-SS-Biotin (21331), streptavidin Sepharose High Performance (17-5113-01), NuPage 4-12% Bis Tris gels, SeeBlue Plus2, and MesNa (sodium-2-mercaptoethanesulfonate) were from Thermo Fisher Scientific.

    DNA Methylation Assay:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: MeDip-chip The global DNA methylation profiles of cells treated with sorafenib or left untreated were obtained by methylated DNA immunoprecipitation coupled with Affymetrix Human Promoter 1.0R Tiling Arrays (MeDip-chip) using a modification of the Affymetrix chromatin immunoprecipitation assay protocol. .. The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol.

    FLAG-tag:

    Article Title: Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity
    Article Snippet: Alexa Fluor (AF)-conjugated secondary IgG antibodies, Transferrin-AF488 (T13342), streptavidin-AF-488 (S32354), immunoprecipitation Kit-Dynabeads® Protein G (10007D), Cholera toxin-subunit B-AF-488 (C34775), biotin EZ-Link Sulfo-NHS-SS-Biotin (21331), streptavidin Sepharose High Performance (17-5113-01), NuPage 4-12% Bis Tris gels, SeeBlue Plus2, and MesNa (sodium-2-mercaptoethanesulfonate) were from Thermo Fisher Scientific. .. Protein G agarose beads (sc-2002) was from Santa Cruz Biotechnology, and Myc-αDDK-tagged (FLAG-tag) ORF clone of Homo sapiens Ca9 (RC204839) and rabbit anti-αDDK antibody (TA100023) were from Origene.

    Staining:

    Article Title: Sorafenib induces variations of the DNA methylome in HA22T/VGH human hepatocellular carcinoma-derived cells
    Article Snippet: The antibody-DNA complexes were immunoprecipitated using Dynabeads® Protein G immunoprecipitation kit (Life Technologies) and the methylated enriched DNA was purified by standard phenol/chloroform procedure and precipitated with isopropanol. .. Hybridization on Human Promoter 1.0R array was performed using the reagents contained in GeneChip® Hybridization, Wash, and Stain kit (Affymetrix) using GeneChip hybridization oven 640 (Affymetrix).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Thermo Fisher dynabeads protein g immunoprecipitation kit
    Microtubule-associated protein 1A/B light chain 3B I (LC3B-I) and LC3B-II interact with macropinosomes at the cell membrane. A , To determine whether LC3B localized to Ankfy1-positive sites, cells were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Cells were permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Images are optical Z sections through the middle plane of the cell body, with 10-μm scale bars. Arrowheads indicate examples of Ankfy1-LC3B colocalization at the cell surface in magnified insets of the indicated area. B , EBOV localizes to LC3B-positive structures at the cell surface. HeLa cells were incubated with Ebola virus-like particles (VLPs) or phosphate-buffered saline for 10 minutes, and samples were stained with anti-EBOV glycoprotein antibody, anti-LC3B antibody, and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of VLP-LC3B colocalization in magnified insets of the indicated area. C , To test whether autophagy-associated proteins are a prerequisite for its association with Ankfy1-positive sites, cells treated with indicated siRNAs were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Samples were then permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of colocalization between Ankfy1 and LC3B in magnified insets. D , Both LC3B-I and LC3B-II bind Ankfy1. siRNA-treated HeLa cells were transfected with plasmids expressing myc-LC3B and either EYFP or EYFP-Ankfy1. After 24 hours, proteins were precipitated with <t>Dynabeads/anti–enhanced</t> green fluorescent protein (eGFP) antibody complexes and analyzed by immunoblotting with anti-eGFP (top panels) and anti-myc (lower panels) antibodies. Abbreviation: IP, <t>immunoprecipitation.</t>
    Dynabeads Protein G Immunoprecipitation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dynabeads protein g immunoprecipitation kit/product/Thermo Fisher
    Average 90 stars, based on 420 article reviews
    Price from $9.99 to $1999.99
    dynabeads protein g immunoprecipitation kit - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    99
    Thermo Fisher dynabeads mrna purification kit
    Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the <t>antibody-ribosome-mRNA</t> complex with protein A <t>dynabeads.</t> After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.
    Dynabeads Mrna Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dynabeads mrna purification kit/product/Thermo Fisher
    Average 99 stars, based on 595 article reviews
    Price from $9.99 to $1999.99
    dynabeads mrna purification kit - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    95
    Thermo Fisher coimmunoprecipitation protein g functionalized dynabeads
    Presence of cAMP increases the binding of pRII antibodies to PKA-II holoenzymes. (A–D) SPR analysis using pRII- (A-B) or RRXpS/pT-specific antibodies (C and D) covalently immobilized on sensor chips. Binding of preformed RIIα 2 :Cα 2 (A and C) and RIIβ 2 :Cα 2 holoenzymes (B and D) was assessed in buffer containing 1 mM MgCl 2 or 1 mM CaCl 2 and 0.5 mM ATP. Dissociation of holoenzymes was induced by adding 10 µM cAMP before SPR analysis. (E) <t>Coimmunoprecipitation</t> of RIIβ and Cα using pRII antibodies captured on <t>protein</t> G functionalized beads. Pull-downs of preformed RIIβ 2 :C 2 holoenzymes were performed in the absence or presence of 1 mM ATP or 0.5 and 10 µM cAMP. Representative immunoblots (left) and densitometry results are shown (right). Values are means ± SD; n = 3. IP, immunoprecipitation; RU, resonance units.
    Coimmunoprecipitation Protein G Functionalized Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/coimmunoprecipitation protein g functionalized dynabeads/product/Thermo Fisher
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    coimmunoprecipitation protein g functionalized dynabeads - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    Image Search Results


    Microtubule-associated protein 1A/B light chain 3B I (LC3B-I) and LC3B-II interact with macropinosomes at the cell membrane. A , To determine whether LC3B localized to Ankfy1-positive sites, cells were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Cells were permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Images are optical Z sections through the middle plane of the cell body, with 10-μm scale bars. Arrowheads indicate examples of Ankfy1-LC3B colocalization at the cell surface in magnified insets of the indicated area. B , EBOV localizes to LC3B-positive structures at the cell surface. HeLa cells were incubated with Ebola virus-like particles (VLPs) or phosphate-buffered saline for 10 minutes, and samples were stained with anti-EBOV glycoprotein antibody, anti-LC3B antibody, and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of VLP-LC3B colocalization in magnified insets of the indicated area. C , To test whether autophagy-associated proteins are a prerequisite for its association with Ankfy1-positive sites, cells treated with indicated siRNAs were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Samples were then permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of colocalization between Ankfy1 and LC3B in magnified insets. D , Both LC3B-I and LC3B-II bind Ankfy1. siRNA-treated HeLa cells were transfected with plasmids expressing myc-LC3B and either EYFP or EYFP-Ankfy1. After 24 hours, proteins were precipitated with Dynabeads/anti–enhanced green fluorescent protein (eGFP) antibody complexes and analyzed by immunoblotting with anti-eGFP (top panels) and anti-myc (lower panels) antibodies. Abbreviation: IP, immunoprecipitation.

    Journal: The Journal of Infectious Diseases

    Article Title: Autophagy-Associated Proteins Control Ebola Virus Internalization Into Host Cells

    doi: 10.1093/infdis/jiy294

    Figure Lengend Snippet: Microtubule-associated protein 1A/B light chain 3B I (LC3B-I) and LC3B-II interact with macropinosomes at the cell membrane. A , To determine whether LC3B localized to Ankfy1-positive sites, cells were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Cells were permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Images are optical Z sections through the middle plane of the cell body, with 10-μm scale bars. Arrowheads indicate examples of Ankfy1-LC3B colocalization at the cell surface in magnified insets of the indicated area. B , EBOV localizes to LC3B-positive structures at the cell surface. HeLa cells were incubated with Ebola virus-like particles (VLPs) or phosphate-buffered saline for 10 minutes, and samples were stained with anti-EBOV glycoprotein antibody, anti-LC3B antibody, and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of VLP-LC3B colocalization in magnified insets of the indicated area. C , To test whether autophagy-associated proteins are a prerequisite for its association with Ankfy1-positive sites, cells treated with indicated siRNAs were transfected with plasmids encoding EYFP-Ankfy1 and myc-LC3B. Samples were then permeabilized and stained with anti-myc antibody (red) and CellMask dye (gray). Optical Z sections through the middle of the cell are shown with 10-μm scale bars. Arrowheads indicate examples of colocalization between Ankfy1 and LC3B in magnified insets. D , Both LC3B-I and LC3B-II bind Ankfy1. siRNA-treated HeLa cells were transfected with plasmids expressing myc-LC3B and either EYFP or EYFP-Ankfy1. After 24 hours, proteins were precipitated with Dynabeads/anti–enhanced green fluorescent protein (eGFP) antibody complexes and analyzed by immunoblotting with anti-eGFP (top panels) and anti-myc (lower panels) antibodies. Abbreviation: IP, immunoprecipitation.

    Article Snippet: Proteins were precipitated using a rabbit anti-eGFP antibody and Dynabeads protein G immunoprecipitation kit (Thermo Fisher) according to the manufacturer’s protocol.

    Techniques: Transfection, Staining, Incubation, Expressing, Immunoprecipitation

    β-1,3-glucan is required for NKp30-mediated killing of Cryptococcus . a Flow cytometric analysis of cryptococcal cell wall/membrane (CCW/M) binding to YT cells as detected by mAb to β-1,3-glucan vs. isotype control Ab. b Flow cytometric analysis of β-glucan binding to YT cell. YT cells were incubated with (left panel) or without (right panel) laminarin, a linear β-1,3-glucan with β-1,6-linkages. c Flow cytometric analysis of laminarihexaose binding to YT cells. YT cells were incubated with laminarihexaose (from curdlan, comprising only β-1,3-glucan). d Comparison of binding of pustulan vs. β-1,3-glucan to YT cells analyzed using flow cytometry. YT cells were incubated with pustulan (comprising only β-1,6-glucan) or β-1,3-glucan (derived from S. cerevisiae ). This experiment was performed twice. e Flow cytometric analysis of a recombinant NKp30-Fc fusion protein binding to β-glucan-conjugated beads (β-1,3-GB) compared to unconjugated polystyrene beads as control. NKp30-Fc on the beads was detected with anti-NKp30 antibody (1C01). f Anti-NKp30 mAb (1C01) blocked NKp30 binding to β-1,3-GB. Recombinant NKp30 was incubated with 1C01 before being applied to beads conjugated with β-1,3-glucan. The presence of NKp30 on β-1,3-GB was detected using the polyclonal anti-NKp30 antibody. g NKp30 binding to C. neoformans vs. C. albicans analyzed using flow cytometry. The experiment was performed twice. h Immunoprecipitation of NKp30 with β-1,3-glucan. YT cell lysate was incubated with β-1,3-glucan (laminarin) before being incubated with protein G beads that had been conjugated with a mAb against β-1,3-glucan. i YT cell killing of C. neoformans ). **, p

    Journal: Nature Communications

    Article Title: Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida

    doi: 10.1038/s41467-018-03014-4

    Figure Lengend Snippet: β-1,3-glucan is required for NKp30-mediated killing of Cryptococcus . a Flow cytometric analysis of cryptococcal cell wall/membrane (CCW/M) binding to YT cells as detected by mAb to β-1,3-glucan vs. isotype control Ab. b Flow cytometric analysis of β-glucan binding to YT cell. YT cells were incubated with (left panel) or without (right panel) laminarin, a linear β-1,3-glucan with β-1,6-linkages. c Flow cytometric analysis of laminarihexaose binding to YT cells. YT cells were incubated with laminarihexaose (from curdlan, comprising only β-1,3-glucan). d Comparison of binding of pustulan vs. β-1,3-glucan to YT cells analyzed using flow cytometry. YT cells were incubated with pustulan (comprising only β-1,6-glucan) or β-1,3-glucan (derived from S. cerevisiae ). This experiment was performed twice. e Flow cytometric analysis of a recombinant NKp30-Fc fusion protein binding to β-glucan-conjugated beads (β-1,3-GB) compared to unconjugated polystyrene beads as control. NKp30-Fc on the beads was detected with anti-NKp30 antibody (1C01). f Anti-NKp30 mAb (1C01) blocked NKp30 binding to β-1,3-GB. Recombinant NKp30 was incubated with 1C01 before being applied to beads conjugated with β-1,3-glucan. The presence of NKp30 on β-1,3-GB was detected using the polyclonal anti-NKp30 antibody. g NKp30 binding to C. neoformans vs. C. albicans analyzed using flow cytometry. The experiment was performed twice. h Immunoprecipitation of NKp30 with β-1,3-glucan. YT cell lysate was incubated with β-1,3-glucan (laminarin) before being incubated with protein G beads that had been conjugated with a mAb against β-1,3-glucan. i YT cell killing of C. neoformans ). **, p

    Article Snippet: Protein G beads (Dynabeads 100.07D, Thermo Fisher) were used to immunoprecipitate NKp30.

    Techniques: Flow Cytometry, Binding Assay, Incubation, Cytometry, Derivative Assay, Recombinant, Protein Binding, Immunoprecipitation

    Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the antibody-ribosome-mRNA complex with protein A dynabeads. After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.

    Journal: eLife

    Article Title: FMRP has a cell-type-specific role in CA1 pyramidal neurons to regulate autism-related transcripts and circadian memory

    doi: 10.7554/eLife.46919

    Figure Lengend Snippet: Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the antibody-ribosome-mRNA complex with protein A dynabeads. After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.

    Article Snippet: Sequencing libraries were prepared using Poly(A) RNA selection (Dynabeads mRNA Purification Kit, ThermoFisher Scientific) and the TruSeq RNA Library Prep Kit (Illumina).

    Techniques: Immunoprecipitation, Titration, Incubation, Protein Extraction, Isolation, Protein Concentration, Western Blot, Concentration Assay

    Presence of cAMP increases the binding of pRII antibodies to PKA-II holoenzymes. (A–D) SPR analysis using pRII- (A-B) or RRXpS/pT-specific antibodies (C and D) covalently immobilized on sensor chips. Binding of preformed RIIα 2 :Cα 2 (A and C) and RIIβ 2 :Cα 2 holoenzymes (B and D) was assessed in buffer containing 1 mM MgCl 2 or 1 mM CaCl 2 and 0.5 mM ATP. Dissociation of holoenzymes was induced by adding 10 µM cAMP before SPR analysis. (E) Coimmunoprecipitation of RIIβ and Cα using pRII antibodies captured on protein G functionalized beads. Pull-downs of preformed RIIβ 2 :C 2 holoenzymes were performed in the absence or presence of 1 mM ATP or 0.5 and 10 µM cAMP. Representative immunoblots (left) and densitometry results are shown (right). Values are means ± SD; n = 3. IP, immunoprecipitation; RU, resonance units.

    Journal: The Journal of Cell Biology

    Article Title: PKA-RII subunit phosphorylation precedes activation by cAMP and regulates activity termination

    doi: 10.1083/jcb.201708053

    Figure Lengend Snippet: Presence of cAMP increases the binding of pRII antibodies to PKA-II holoenzymes. (A–D) SPR analysis using pRII- (A-B) or RRXpS/pT-specific antibodies (C and D) covalently immobilized on sensor chips. Binding of preformed RIIα 2 :Cα 2 (A and C) and RIIβ 2 :Cα 2 holoenzymes (B and D) was assessed in buffer containing 1 mM MgCl 2 or 1 mM CaCl 2 and 0.5 mM ATP. Dissociation of holoenzymes was induced by adding 10 µM cAMP before SPR analysis. (E) Coimmunoprecipitation of RIIβ and Cα using pRII antibodies captured on protein G functionalized beads. Pull-downs of preformed RIIβ 2 :C 2 holoenzymes were performed in the absence or presence of 1 mM ATP or 0.5 and 10 µM cAMP. Representative immunoblots (left) and densitometry results are shown (right). Values are means ± SD; n = 3. IP, immunoprecipitation; RU, resonance units.

    Article Snippet: Coimmunoprecipitation Protein G functionalized Dynabeads (Thermo Fisher Scientific) were used to probe binding of PKA-II holoenzymes to the phospho-RII antibodies.

    Techniques: Binding Assay, SPR Assay, Western Blot, Immunoprecipitation