A32965, by Thermo Fisher Scientific - Product details

1

Purification of His-tagged INTS10 from HEK293T

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Human expression plasmids encoding codon-optimized INTS10 or His8-INTS10 were synthesized (Twist Bioscience) and transfected into HEK 293T/17 cells (ATCC CRL-11268) with FuGene HD (Promega E2311) according to the manufacturer’s protocol. Two days later, the cells were washed twice with DPBS and harvested with IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol; ThermoFisher 87787) supplemented with protease inhibitors (ThermoFisher A32965). Lysates were nutated at 4° for 30 mins, clarified by centrifugation at 12,000xg for 10 minutes, and snap-frozen. Concentrations were measured with the BCA assay (ThermoFisher 23225).
Lysates were thawed on ice, supplemented with imidazole to 10 mM, and nutated at 4° for 30 minutes with cobalt magnetic beads (ThermoFisher 10103D) pre-equilibrated in IP lysis buffer + 10 mM imidazole. The beads were separated on a magnet, washed twice with lysis buffer + 10 mM imidazole, and eluted with lysis buffer + 300 mM imidazole.

Replogle J.M., Saunders R.A., Pogson A.N., Hussmann J.A., Lenail A., Guna A., Mascibroda L., Wagner E.J., Adelman K., Lithwick-Yanai G., Iremadze N., Oberstrass F., Lipson D., Bonnar J.L., Jost M., Norman T.M, & Weissman J.S. (2022). Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell, 185(14), 2559-2575.e28.

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2

Co-immunoprecipitation of protein complexes

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Cells from a 10-cm plate were washed with 1× PBS and lysed with 1 ml of co-IP buffer (50 mM Tris–Cl [pH 7.4], 150 mM NaCl, 15 mM MgCl₂, 5 mM EDTA, and 0.1% Nonidet P-40), containing a cocktail of protease inhibitors (A32965; Thermo Fisher Scientific). Cell extracts were briefly sonicated and centrifuged at 21 000 × g for 10 min to remove insoluble debris. Cell lysates were incubated with antibodies overnight at 4°C, followed by incubation with Protein A/G polyacrylamide beads (53133; Thermo Fisher Scientific) for 2 h. Beads were then washed three times with co-IP buffer, and bound proteins were eluted by SDS-PAGE loading buffer. Proteins were resolved on an SDS-PAGE gel and analyzed by western blot analysis.

Yuan W., Al-Hadid Q., Wang Z., Shen L., Cho H., Wu X, & Yang Y. (2021). TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution. Nucleic Acids Research, 49(15), 8573-8591.

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3

Detecting Protein Expression in Lung Tissue

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For detection of protein expression in lung tissue, samples were lysed in a 1% SDS buffer (1% SDS, 50mM triethanolamine pH 7.4, 150mM NaCl) containing a cocktail of phosphatase protease inhibitors (Sigma, 4906845001) and protease inhibitors (Thermo Scientific, A32965). The lysates were centrifuged at 15,000 rpm for 10 min and soluble protein supernatents were used for Western blot analysis. Equal amounts of protein (30ug) were separated by SDS-PAGE and transferred onto membranes. Membranes were blocked with 10% non-fat milk in Phosphate-buffered saline with 0.1% Tween-20 (PBST) and probed with antibodies against GSDMD (abcam, ab219800) and actin (abcam, ab3280). For Western blotting of THP-1 cells, samples were lysed in 1% SDS containing protease inhibitors prior to SDS-PAGE separation as described above. Membranes were probed with antibodies against influenza virus nucleoprotein (abcam, ab20343), GSDMD (abcam, ab210070), and GAPDH (Thermo Scientific, ZG003).

Speaks S., Zani A., Solstad A., Kenney A., McFadden M.I., Zhang L., Eddy A.C., Amer A.O., Robinson R., Cai C., Ma J., Hemann E.A., Forero A, & Yount J.S. (2023). Gasdermin D promotes influenza virus-induced mortality through neutrophil amplification of inflammation. bioRxiv.

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4

Immunoprecipitation and Western Blot Analysis

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RIPA lysis buffer containing 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS was used for WB, and a lysis buffer containing 1% Nonidet P-40 and 5% glycerol was used for coIP. Both buffers were supplemented with PMSF, NaF, Na3VO4, and protease inhibitors (A32965; Thermo Fisher Scientific). For coIP, 300-500 μg of the total protein, 1-2 μg of a primary antibody, and 30 μL of slurry of protein G Sepharose 4 Fast Flow (GE Healthcare Life Sciences, Pittsburgh, PA) were mixed and rotated at 4°C for overnight. After washing the Sepharose beads for four times, Laemmli sample buffer (Bio-Rad, Hercules, CA) was added into elute the precipitated proteins for WB. WB was performed as previously described^{1 (link),28 ,33 (link)} and densitometric analysis was performed using the Image J software (NIH).

Liu D., Fu X., Wang Y., Wang X., Wang H., Wen J, & Kang N. (2020). Protein diaphanous homolog 1 (Diaph1) promotes myofibroblastic activation of hepatic stellate cells by regulating Rab5a activity and TGFβ receptor endocytosis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(6), 7345-7359.

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5

Quantifying Human Aβ42 in 5XFAD Mouse Cortex

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Cortical tissue was harvested at 49 weeks of age, and prepared according to the protocol established for 5XFAD mice by Oakley, et al.^{10 (link)}. Specifically, following extraction, the tissue was flash frozen and re-suspended in 3 to 4 volumes of PBS-0.5% Triton supplemented with protease inhibitors (A32965 ThermoFisher, Waltham, MA). Tissue was homogenized with a Corning Dounce homogenizer (1234F35 Thomas Scientific, Swedesboro, NJ), freeze–thawed 3 times, and cleared at 15,000 rpm for 15 min at 4 °C. Cleared homogenates were supplemented with guanidine hydrochloride to a final concentration of 5 M to solubilize plaques. To detect human Aβ42 levels, cleared homogenates were diluted in Standard Diluent Buffer and measured by the Invitrogen Human Aβ42 ELISA kit (KHB3441, ThermoFisher, Waltham, MA) following manufacturer’s instructions. Final guanidine hydrochloride concentrations were under 0.1 M. All samples were run in duplicate; their readings fell within the range of the standard dose responses curve. Total protein was measured by a protein assay kit (5000002, Bio-Rad, Hercules, CA) and used for normalization.

Nagare R., Possidente B., Lagalwar S, & Figueiro M.G. (2020). Robust light–dark patterns and reduced amyloid load in an Alzheimer’s disease transgenic mouse model. Scientific Reports, 10, 11436.

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6

Purification of His-tagged INTS10 from HEK293T

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Human expression plasmids encoding codon-optimized INTS10 or His8-INTS10 were synthesized (Twist Bioscience) and transfected into HEK 293T/17 cells (ATCC CRL-11268) with FuGene HD (Promega E2311) according to the manufacturer’s protocol. Two days later, the cells were washed twice with DPBS and harvested with IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol; ThermoFisher 87787) supplemented with protease inhibitors (ThermoFisher A32965). Lysates were nutated at 4° for 30 mins, clarified by centrifugation at 12,000xg for 10 minutes, and snap-frozen. Concentrations were measured with the BCA assay (ThermoFisher 23225).
Lysates were thawed on ice, supplemented with imidazole to 10 mM, and nutated at 4° for 30 minutes with cobalt magnetic beads (ThermoFisher 10103D) pre-equilibrated in IP lysis buffer + 10 mM imidazole. The beads were separated on a magnet, washed twice with lysis buffer + 10 mM imidazole, and eluted with lysis buffer + 300 mM imidazole.

Replogle J.M., Saunders R.A., Pogson A.N., Hussmann J.A., Lenail A., Guna A., Mascibroda L., Wagner E.J., Adelman K., Lithwick-Yanai G., Iremadze N., Oberstrass F., Lipson D., Bonnar J.L., Jost M., Norman T.M, & Weissman J.S. (2022). Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell, 185(14), 2559-2575.e28.

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7

Western Blot Analysis of Testicular Proteins

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Mouse testes were collected and homogenized in RIPA buffer (J63306-AP, Thermo Fisher Scientific) with protease inhibitor (A32965, Thermo Fisher Scientific). Protein lysates were separated by 4–20% polyacrylamide gels (4561096, Bio-Rad) and transferred to PVDF membranes. The membranes were blocked in 5% non-fat milk at RT for 30 min and subsequently incubated with primary antibodies in 5% non-fat milk overnight at 4 °C. The primary antibodies used were anti-MIWI (1:1000; 2079, Cell Signaling Technology), anti-MILI (1:2000; PM044, MBL) anti-TDRKH (1:4000; 13528–1-AP, Proteintech), or HRP-conjugated mouse anti-β-actin (1:5000; A3854, Sigma-Aldrich). Membranes were washed with TBST and incubated with HRP-conjugated goat anti-rabbit IgG (1:5000; 1706515, Bio-Rad) at RT for 1 h followed by chemiluminescent detection with ECL Substrate (1705060, Bio-Rad).

Wei C., Yan X., Mann J.M., Geng R., Xie H., Demireva E.Y., Sun L., Ding D, & Chen C. (2023). PNLDC1 catalysis and postnatal germline function are required for piRNA trimming, LINE1 silencing, and spermatogenesis in mice. bioRxiv.

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8

Immunoprecipitation and Western Blotting of Flag-Tagged Proteins

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Cells were collected in RIPA lysis buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0, 1% SDS, 1 mM EDTA pH 8, 1 mM PMSF, 0, 5% Sodium Deoxycholate) supplemented with protease inhibitors (Thermo Scientific A32965) and incubated 30 min on ice. After centrifuging at 500 g at 4°C, supernatants were quantified. One milligram (1 mg) of total protein from cell lysate was incubated with 40 μl of Anti-Flag M2 Affinity Gel (Sigma-Aldrich) overnight at 4°C. Then, beads were centrifuged and washed with PBS three times for 10 min at 4°C and then resuspended in 50 μl of resuspension buffer (50 mM Tris/HCl pH 7.4, 5 mM EDTA, 10 mM DTT, 1% SDS) plus 20 μl of SDS 5×. Western blot analysis was performed by using anti-Flag (Sigma-Aldrich, M2), anti-Hsc70 (Thermo Scientific, 13D3) and anti-Lamp2A (Abcam ab18528).

Ormeño F., Hormazabal J., Moreno J., Riquelme F., Rios J., Criollo A., Albornoz A., Alfaro I.E, & Budini M. (2020). Chaperone Mediated Autophagy Degrades TDP-43 Protein and Is Affected by TDP-43 Aggregation. Frontiers in Molecular Neuroscience, 13, 19.

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9

Immunoprecipitation and Western Blot Analysis

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RIPA lysis buffer containing 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS was used for WB, and a lysis buffer containing 1% Nonidet P-40 and 5% glycerol was used for coIP. Both buffers were supplemented with PMSF, NaF, Na3VO4, and protease inhibitors (A32965; Thermo Fisher Scientific). For coIP, 300-500 μg of the total protein, 1-2 μg of a primary antibody, and 30 μL of slurry of protein G Sepharose 4 Fast Flow (GE Healthcare Life Sciences, Pittsburgh, PA) were mixed and rotated at 4°C for overnight. After washing the Sepharose beads for four times, Laemmli sample buffer (Bio-Rad, Hercules, CA) was added into elute the precipitated proteins for WB. WB was performed as previously described^{1 (link),28 ,33 (link)} and densitometric analysis was performed using the Image J software (NIH).

Liu D., Fu X., Wang Y., Wang X., Wang H., Wen J, & Kang N. (2020). Protein diaphanous homolog 1 (Diaph1) promotes myofibroblastic activation of hepatic stellate cells by regulating Rab5a activity and TGFβ receptor endocytosis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(6), 7345-7359.

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10

Immunoprecipitation of MIWI and MILI from Mouse Testes

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Mouse testes were collected and homogenized using lysis buffer (20 mM HEPES pH 7.3, 150 mM NaCl, 2.5 mM MgCl₂, 0.2 % NP-40, and 1 mM DTT) with protease inhibitor (A32965, Thermo Fisher Scientific) and RNase inhibitor (N2615, Promega). The lysates were pre-cleared using Protein A agarose beads (11134515001, Sigma-Aldrich) at 4 °C for 2 h. Anti-MIWI (2079, Cell Signaling Technology) or anti-MILI (PM044, MBL) antibody together with Protein A agarose beads were added to the lysates and incubated at 4 °C for 4 h. The beads were washed in lysis buffer 5 times. Immunoprecipitated RNAs were isolated from the beads using Trizol reagent (15596026, Thermo Fisher Scientific) for piRNA labeling or small RNA library construction. For protein detection, immunoprecipitated beads were boiled in protein loading buffer for 5 min. Western blotting of MIWI or MILI was performed as described above.

Wei C., Yan X., Mann J.M., Geng R., Xie H., Demireva E.Y., Sun L., Ding D, & Chen C. (2023). PNLDC1 catalysis and postnatal germline function are required for piRNA trimming, LINE1 silencing, and spermatogenesis in mice. bioRxiv.

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A32965

Lab products found in correlation

14 protocols using a32965

Purification of His-tagged INTS10 from HEK293T

Co-immunoprecipitation of protein complexes

Detecting Protein Expression in Lung Tissue

Immunoprecipitation and Western Blot Analysis

Quantifying Human Aβ42 in 5XFAD Mouse Cortex

Purification of His-tagged INTS10 from HEK293T

Western Blot Analysis of Testicular Proteins

Immunoprecipitation and Western Blotting of Flag-Tagged Proteins

Immunoprecipitation and Western Blot Analysis

Immunoprecipitation of MIWI and MILI from Mouse Testes

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