dynabead protein a, by Thermo Fisher Scientific - Product details

1

RIPK1-Mediated Necroptosis Signaling

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Stimulated T cells were incubated in lysis buffer. For signaling complex analyses, cell lysates were pre-cleared with Protein G beads prior to immunoprecipitation with anti-RIPK1 antibody. Antibodies and reagents used for immunoprecipitation and immunoblotting studies included: anti-RIPK1, anti-Bim, anti-Bclx, (BD Biosciences), nec1, anti-Bcl2, β-actin (Merck chemicals), anti-RIPK3 (ProSci Incorporated), anti-ubiquitin, anti-Bax, anti-RIPK3 (Santa Cruz Biotechnology), anti-RIPK1, anti-A20 (Cell signaling Technology), QVD, ZVAD (Enzo Life Science).
Signaling studies in MEFs were performed as described^{19 (link)}. RIPK1 immunoprecipitations were performed using anti-RIPK1 (Cell Signaling) and Dynabeads M270 Epoxy (Invitrogen). K63-ubiquitin immunoprecipitations were performed using anti-K63 (Millipore) and Dynabead Protein A (Invitrogen). Studies of RIPK3 mutants in A20^−/−Ripk3^−/− MEFs were performed by generating RIPK3 lysine mutants, introducing mutant RIPK3-encoding cDNAs into GFP expressing lentiviral constructs, and infecting A20^−/−Ripk3^−/− MEFs with these viruses. Productively infected cells were FACS-sorted to obtain pure populations of RIPK3-expressing cells prior to being tested in necroptosis assays. Cell survival of MEFs was quantitated using the CellTiter-Glo Luminescent Cell Viability Assay per manufacturer’s instructions (Promega).

Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.

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2

ChIP-qPCR Analysis of Macrophage Chromatin

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Macrophages plated in tissue culture dishes were fixed with formaldehyde and the fixing reaction stopped with glycine. Fixed cells were lysed and sonicated at the high power output for 20 cycles with the Bioruptor (Diagenode). The samples were then incubated with antibody conjugated to Dynabead Protein A (Invitrogen, cat#: 100-02D) at 4°C overnight. The DNA fragments were purified with the Qiaquick PCR purification kit (Qiagen, cat#: 28106) and analyzed by quantitative real-time PCR. Their enrichment was normalized relative to input DNA amount. Primer sequences are as following:
CXCL10 promoter forward: 5’-GTGCTGAGACTGGAGGTTCC-3’; reverse: 5’-GGGAGGGAAAATGGCTTTGC-3’. CXCL10 enhancer forward: 5’-CCGTTTCAGTCGCTATTGATTT-3’; reverse: 5’-CTGATGTCCTCCTGCTCACTTT-3’. HBB promoter forward: 5’-GAGGGCTGAGGGTTTGAAGT-3’; reverse: 5’-TGCTCCTGGGAGTAGATTGG-3’.

Chan C.H., Fang C., Yarilina A., Prinjha R.K., Qiao Y, & Ivashkiv L.B. (2014). BET Bromodomain Inhibition Suppresses Transcriptional Responses to Cytokine-Jak-STAT Signaling in a Gene-specific Manner in Human Monocytes. European journal of immunology, 45(1), 287-297.

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3

Immunoprecipitation of Pf-Hsp60 and Pf-calpain

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The eluted proteins and parasite-infected RBCs were resuspended in native lysis buffer, i.e., 20 mM Tris HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40 (NP-40), 2 mM EDTA, and a 500 µg protein was mixed with 10 µg of anti-Pf-Hsp60 for overnight at 4˚C on a rocking platform. Next day, 50 µl of Dynabead Protein A (Invitrogen, Carlsbad, California, USA) were added to antigen-antibody complex, and this binding was allowed to proceed for 4 hr. Finally, the immunoprecipitate was then collected by adding 0.1 M citrate (pH 2.5) to antigen-antibody bead complex for 2 min. SDS-PAGE was performed, and the protein was transferred onto a PVDF membrane. The blot was analysed with anti-Pf-calpain (1:100 dilution) and anti-Pf-Hsp60 (1:2,000 dilution). Negative control sample was prepared with anti-rabbit IgG for specificity of immunoprecipitation.

Yeo S.J., Liu D.X, & Park H. (2015). Potential Interaction of Plasmodium falciparum Hsp60 and Calpain. The Korean Journal of Parasitology, 53(6), 665-673.

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4

Quantification of siPCSK9 Ago Association

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A mixture of 50 nM NT with 50 nM siPCSK-A1 or of 50 nM NT with 50 nM siPCSK9-A1-6pi was transfected into HeLa cells (n=3) using Lipofectamine 2000 (Invitrogen). After 24 h incubation, the transfected cells were collected to immunoprecipitate the Ago complex. Briefly, the cells were lysed in lysis buffer (1 × PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40, protease inhibitor cocktail (Roche), RNAsin (Promega)) and by Bioruptor (Diagenode). The DNA in the lysates was degraded by RQ1 DNase at 37 °C for 5 min. Immunoprecipitation of Ago complex was performed by 2E12 (Abnova) and Dynabead Protein A (Invitrogen) for 4 h at 4 °C. Small RNAs in the complex were then tailed with adenosines using the Poly(A) tailing kit (Ambion) and used to generate cDNA with Superscript III (Invitrogen) and oligo(dT) adaptor primers. The resulting cDNAs were amplified as described in the small RNA qPCR method for quantification of siPCSK9-A1 or siPCS9-A1-6pi associated with Ago (normalized to Ago-associated control NT).

Lee H.S., Seok H., Lee D.H., Ham J., Lee W., Youm E.M., Yoo J.S., Lee Y.S., Jang E.S, & Chi S.W. (2015). Abasic pivot substitution harnesses target specificity of RNA interference. Nature Communications, 6, 10154.

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5

m6A Immunoprecipitation and RNA Sequencing

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Total RNA was extracted from HCMV-infected HFF (lytic infection), Kasumi-3 (latent infection), and PMA-treated Kasumi-3 (reactivation) cells. Total RNA was treated with recombinant DNase I (Takara) for 1 h at 37 °C and purified using TRIzol LS (Invitrogen). Poly(A) RNA enrichment was performed with 150 μg of total RNA using a Poly(A) Purist Mag kit (Invitrogen), followed by ethanol precipitation. Eluted RNA was incubated with 1.5 μg of anti-m⁶A antibody (Merck, ABE572) in MeRIP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 0.1% NP-40) for 2 h at 4 °C. The immunoprecipitation mixture was mixed with 1 mg of Dynabead protein A (Invitrogen) and incubated overnight at 4 °C. Beads were then washed five times with MeRIP buffer, and RNA was eluted twice by incubation in MeRIP buffer containing 6.7 mM m⁶A sodium salt (Sigma) for 1 h at 4 °C. Eluted and bead-bound RNA were purified using the NucleoSpin RNA Clean-up kit (Macherey–Nagel) and subjected to RT-PCR.

Lee S., Kim H., Hong A., Song J., Lee S., Kim M., Hwang S.Y., Jeong D., Kim J., Son A., Lee Y.S., Kim V.N., Kim J.S., Chang H, & Ahn K. (2022). Functional and molecular dissection of HCMV long non-coding RNAs. Scientific Reports, 12, 19303.

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6

RIPK1-Mediated Necroptosis Signaling

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Stimulated T cells were incubated in lysis buffer. For signaling complex analyses, cell lysates were pre-cleared with Protein G beads prior to immunoprecipitation with anti-RIPK1 antibody. Antibodies and reagents used for immunoprecipitation and immunoblotting studies included: anti-RIPK1, anti-Bim, anti-Bclx, (BD Biosciences), nec1, anti-Bcl2, β-actin (Merck chemicals), anti-RIPK3 (ProSci Incorporated), anti-ubiquitin, anti-Bax, anti-RIPK3 (Santa Cruz Biotechnology), anti-RIPK1, anti-A20 (Cell signaling Technology), QVD, ZVAD (Enzo Life Science).
Signaling studies in MEFs were performed as described^{19 (link)}. RIPK1 immunoprecipitations were performed using anti-RIPK1 (Cell Signaling) and Dynabeads M270 Epoxy (Invitrogen). K63-ubiquitin immunoprecipitations were performed using anti-K63 (Millipore) and Dynabead Protein A (Invitrogen). Studies of RIPK3 mutants in A20^−/−Ripk3^−/− MEFs were performed by generating RIPK3 lysine mutants, introducing mutant RIPK3-encoding cDNAs into GFP expressing lentiviral constructs, and infecting A20^−/−Ripk3^−/− MEFs with these viruses. Productively infected cells were FACS-sorted to obtain pure populations of RIPK3-expressing cells prior to being tested in necroptosis assays. Cell survival of MEFs was quantitated using the CellTiter-Glo Luminescent Cell Viability Assay per manufacturer’s instructions (Promega).

Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.

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7

ChIP-qPCR Profiling of STAT1/3 Binding

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In brief, 1 × 10⁷ A549 cells were treated with 20 ng/mL of IFNγ for 1.5 h or sequentially treated with 20 ng/mL of IFNγ for 2 h with 20 ng/mL of EGF for 1.5 h. After treatment, A549 cells were fixed by 1% formaldehyde and fragmentized by sonication. A549 cells were resuspended and incubated with control IgG, anti-STAT3, and anti-STAT1 antibodies (Cell Signaling, Danvers, MA, USA) at 4 °C overnight for immunoprecipitation. After incubation, the antibodies were captured by Dynabead-Protein A (Life Technologies, Waltham, MA, USA). The Dynabeads were washed and consequently pulled down by a Sample Magnetic Rack. DNA fragments were eluted by boiling the Dynabeads and concentrated by a FavorPrep GEL/PCR Purification Mini Kit (Favorgen Biotech Corp., Wembley, WA, Australia). The DNA preparation was analyzed by real-time PCR using Fast SYBR Green Master Mix (Applied Biosystem, CA, USA) with primer pairs shown in Table 1. The primer sequences amplify the PDL1 and MCL1 promoter predicted from PROMO (http://alggen.lsi.upc.es/, accessed on 4 May 2021).

Wang C.I., Chang Y.F., Sie Z.L., Ho A.S., Chang J.S., Peng C.L, & Cheng C.C. (2021). Irradiation Suppresses IFNγ-Mediated PD-L1 and MCL1 Expression in EGFR-Positive Lung Cancer to Augment CD8+ T Cells Cytotoxicity. Cells, 10(10), 2515.

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8

Isolation and Detection of IFNα and IFNγ

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2 × 10⁶ A549 cells in a 6-well plate were treated with 0 and 10 Gy irradiation and cultured at 37 °C for 24 h. The culture medium was collected and centrifuged at 2000 rpm for 10 min to remove cell debris. The supernatant was consequently incubated with 1 μg/mL of rabbit-anti-IFNα (Elabscience, Houston, Texas, USA) and rabbit-anti-IFNγ antibodies (ABclonal, Woburn, MA, USA) at 4 °C overnight. The antibodies were consequently captured by 0.2 mg/mL of Dynabead-Protein A (Life Technologies, Waltham, Massachusetts, USA) at room temperature for 2 h. The Dynabeads were pulled down by a Sample Magnetic Rack and the supernatant was collected. To make sure that IFNα and IFNγ were expressed in the medium, Western blots were used for detecting IFNα and IFNγ. In brief, Dynabeads were washed with 1 mL of PBS buffer and resuspended in 100 μL of RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5% sodium deoxycholate, 1% IGEPAL, and 0.1% SDS) with 20 μL of sixfold SDS sample buffer (Alfa Aesar, Heysham, Lancashire, UK). The same antibodies targeting IFNα and IFNγ were used to detect the targets in Western blots.

Wang C.L., Ho A.S., Chang C.C., Sie Z.L., Peng C.L., Chang J, & Cheng C.C. (2023). Radiotherapy enhances CXCR3highCD8+ T cell activation through inducing IFNγ-mediated CXCL10 and ICAM-1 expression in lung cancer cells. Cancer Immunology, Immunotherapy, 72(6), 1865-1880.

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9

NCBP2 Ribonucleoprotein Immunoprecipitation

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~2 × 10⁷ cells were harvested and washed with cold PBS twice and lysed in 1 ml RIP-lysis buffer [200 mM NaCl, 50 mM Tris pH 8, 1 mM EDTA, 0.5% IGEPAL CA-630 (Sigma I8896), 100 U ml⁻¹ SUPERase-in RNase inhibitor (Invitrogen AM2694), 1X Complete Mini EDTA-free protease inhibitor (Sigma 4693159001), 1X Phosphatase inhibitor cocktail 2 (Sigma P5726)] for 30 min on ice, with 10 s vortex-mixing every 10 min. Lysed cells were centrifuged at 16,000 × g at 4 °C for 30 min and the supernatant was collected as cell lysate. 1 mg of cell lysate was then mixed with 10 µg rabbit anti-NCBP2 antibody (Abcam ab91560) or 10 µg of rabbit IgG (Abcam ab37415) as a mock-RIP. Antibody-lysate mixture was tumbled at 4 °C for 2 h. 50 µl Dynabead-proteinA (Life Technologies 10002D) was washed six times with 1 ml cold RIP-wash buffer [150 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA, 0.1% IGEPAL CA-630, 1X Complete Mini EDTA-free protease inhibitor] and mixed with the antibody-lysate mixture before being mixed by tumbling at 4 °C for 2 h. Beads were washed four times with 0.5 ml cold RIP-wash buffer with a single change of tube. Beads were either boiled in 40 µl 1X Laemmli buffer for western blotting or incubated in Trizol-LS (Ambion 10296) for RNA isolation.

Koh C.W., Goh Y.T, & Goh W.S. (2019). Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes. Nature Communications, 10, 5636.

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10

Immunopurification of Protein Complexes

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Cells were harvested in immunopurification buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton™ X-100 with protease and phosphatase inhibitor). After centrifuging (14 000 rpm for 30 s at 4°C), the supernatant was collected and incubated with immunopurification antibody and Dynabead protein A (Life Technologies) overnight at 4°C. The Dynabead® protein A-antibody-protein complex was purified using magnetic separation and washed with immunopurification buffer before eluted in loading buffer.

Chen H.J., Mitchell J.C., Novoselov S., Miller J., Nishimura A.L., Scotter E.L., Vance C.A., Cheetham M.E, & Shaw C.E. (2016). The heat shock response plays an important role in TDP-43 clearance: evidence for dysfunction in amyotrophic lateral sclerosis. Brain, 139(5), 1417-1432.

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Dynabead protein a

Lab products found in correlation

16 protocols using dynabead protein a

RIPK1-Mediated Necroptosis Signaling

ChIP-qPCR Analysis of Macrophage Chromatin

Immunoprecipitation of Pf-Hsp60 and Pf-calpain

Quantification of siPCSK9 Ago Association

m6A Immunoprecipitation and RNA Sequencing

RIPK1-Mediated Necroptosis Signaling

ChIP-qPCR Profiling of STAT1/3 Binding

Isolation and Detection of IFNα and IFNγ

NCBP2 Ribonucleoprotein Immunoprecipitation

Immunopurification of Protein Complexes

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