Pg m3

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The PG-M3 is a laboratory instrument designed for cell growth and cell culture applications. It provides precise temperature and CO2 control to maintain optimal conditions for cell cultures. The core function of the PG-M3 is to provide a stable and regulated environment for the cultivation and growth of various cell types.

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Lab products found in correlation

Rabbit polyclonal pml, by Santa Cruz Biotechnology (1 mentions) Mouse β actin, by Merck Group (1 mentions) Ripa buffer, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Passive lysis buffer (plb), by Promega (1 mentions) Ab53773, by Abcam (1 mentions) Rad50, by Santa Cruz Biotechnology (1 mentions) Ab6046, by Abcam (1 mentions) Anti ubiquitin clone fk2, by Enzo Life Sciences (1 mentions) U5379, by Merck Group (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Anti flag m2, by Merck Group (1 mentions) Alexa fluor 546, by Thermo Fisher Scientific (1 mentions) Tetradecanoyl phorbol acetate, by Merck Group (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) γh2ax 05 636, by Merck Group (1 mentions) Gfp a 11122, by Thermo Fisher Scientific (1 mentions) Phospho rb 780 9307, by Cell Signaling Technology (1 mentions) Atrx a301 045a, by Fortis Life Sciences (1 mentions)

7 protocols using pg m3

Cobalous chloride treatment of NB4 cells

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When indicated, NB4 cells (shCTRL or shHIFs) were treated for 24 h with 100 μM CoCl₂ before lysis. For Western blot, proteins were extracted with RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Roche); for luciferase assays, cells were lyzed in passive lysis buffer (Promega); for co-immunoprecipitation experiments, cells were lyzed after mild cross-linking with 0.4% formaldehyde (7 min at RT) in CO-IP buffer (10 mM NaCl, 10 mM Tris–HCl pH 7.5, 5 mM MgCl₂, 0.5% CHAPS) supplemented with protease and phosphatase inhibitors (Pierce) and then briefly sonicated to extract nuclear proteins. Total lysates were immunoprecipitated with an antibody against PML (PG-M3; Santa Cruz).
Cell extracts and immunoprecipitates were resolved by SDS–PAGE 7.5–10% and transferred to a PVDF membrane (Biorad). Non-specific binding was blocked in 5% non-fat milk for 1 h at RT and blotted with the following antibodies: rabbit polyclonal HIF-1α (Cayman), rabbit polyclonal PML (Santa Cruz) and rabbit polyclonal PML (Novus). Mouse β-actin (Sigma) was used as internal loading control.

Coltella N., Percio S., Valsecchi R., Cuttano R., Guarnerio J., Ponzoni M., Pandolfi P.P., Melillo G., Pattini L, & Bernardi R. (2014). HIF factors cooperate with PML-RARα to promote acute promyelocytic leukemia progression and relapse. EMBO Molecular Medicine, 6(5), 640-650.

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Characterization of BRCA1 and PML

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Antibodies for immunofluorescence included: BRCA1 (A301-378A, Bethyl Laboratories; OP92, Calbiochem), and PML (PG-M3, Santa Cruz; ab53773, Abcam). Antibodies for western blot included: BRCA1, RAD50 (Santa Cruz), TRF2 (Imgenex) and lamin B (C-20, Santa Cruz). The BLM antibody was a generous gift from Dr. Albert Davalos. BRCA1 siRNA (ON target plus Human BRCA1 (672) siRNA SMARTpool) was purchased from Thermo Scientific.

Acharya S., Kaul Z., Gocha A.S., Martinez A.R., Harris J., Parvin J.D, & Groden J. (2014). Association of BLM and BRCA1 during Telomere Maintenance in ALT Cells. PLoS ONE, 9(8), e103819.

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Antibody Panel for Western Blotting and IF

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Anti-PML for Western blotting and immunofluorescence (PG-M3, Santa Cruz), anti-FLAG (M2, Sigma), anti-β-tubulin (ab6046, Abcam), anti-GAPDH (#2118, Cell Signaling), anti-GFP (D5.1 XP, Cell Signaling), anti-HA for Western blotting (clone 16B12, Covance), anti-ubiquitin (clone FK2, Enzo LifeSciences, and whole antiserum U5379, Sigma), anti-SUMO1 and anti-SUMO2 antisera were prepared in house using mature recombinant SUMO1 and SUMO2 as antigen (7 (link)). Anti-SENP1 and anti-SENP6 were prepared in house using residues 419–643 as antigen for SENP1 and residues 628–1112 for SENP6, followed by affinity purification. SENP5 antiserum was kindly provided by Dr. Heidi McBride, McGill University Montreal Canada (38 (link)).

Fasci D., Anania V.G., Lill J.R, & Salvesen G.S. (2015). SUMO deconjugation is required for arsenic-triggered ubiquitylation of PML. Science signaling, 8(380), ra56.

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Antibody Detection of KSHV Proteins

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A mouse monoclonal (PG-M3) and rabbit polyclonal antibodies (Ab; H-238) against PML were purchased from Santa Cruz Biotechnology and used to detect endogenous and exogenous PML. Rabbit polyclonal anti-hDAXX (HPA008736) and anti-SP100 (HPA016707) Abs were purchased from Sigma-Aldrich. Mouse monoclonal antibodies to RTA, K8 (K-bZIP), K5, K3, K9 (vIRF1), ORF59, and K8.1 were developed previously in our laboratory and rabbit polyclonal Ab against gB was a kind gift from Bala Chandran (Akula et al., 2001 (link); Okuno et al., 2002 (link)). A rat monoclonal anti-LANA (HHV-8 ORF73) Ab was purchased from Advanced Biotechnologies (Cat# 13-210-100). An anti-Halo Tag^® mouse monoclonal Ab (Cat# G9211, Promega, United States) was used to detect exogenous Halo-tagged PML. A mouse monoclonal antibody (Sigma Cat# T5201) was used to detect β-tubulin. Horseradish peroxidase (HRP)-labeled goat polyclonal anti-rabbit, anti-mouse, and anti-rat immunoglobulins (Dako, Denmark) were used as secondary antibodies in Western blot analysis. Anti-mouse, anti-rat, and anti-rabbit IgG conjugated Alexa Fluor^® 488 and/or Alexa Fluor^® 546 (Molecular Probes, United States) were used as secondary antibodies for immunofluorescence assays (IFA). Tetradecanoyl phorbol acetate (TPA; Sigma, Japan) and sodium butyrate (NaB; Sigma, Japan) were used to induce lytic replication of KSHV.

Hossain M.G., Ohsaki E., Honda T, & Ueda K. (2018). Importance of Promyelocytic Leukema Protein (PML) for Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication. Frontiers in Microbiology, 9, 2324.

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Antibody Immunoblotting and Immunofluorescence Panel

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Antibodies were as follows (catalog number and immunoblot diltuions (if applicable) in parentheses): MDM2 (SMP14, 1:500), total Rb (IF8, 1:200), cyclin A (H432, 1:1000), p16 (C20, 1:1000), p53 (DO-1 and Bp53-12, 1:500), tubulin (C20, 1:2000), ATRX (H-300, 1:1000 and D-5, 1:500), PML (PG-M3), and normal rabbit IgG (SC-2027) were obtained from Santa Cruz Biotechnology, phospho-Rb 780 (#9307, 1:1000) from Cell Signaling, HP1γ (05-690) and γH2AX (05-636) from Millipore, ATRX (A301-045A, 1:2000) from Bethyl Laboratories, 53BP1 (ab172580) and LC3 (ab48394) from Abcam, GFP (A11122) from Life Technologies. Dilutions for immunofluorescence were as follows: ATRX (A301-045A) 1:2000; HP1γ (05-690) 1:5000; PML (PG-M3) 1:1000; γH2AX (05-636) 1:200; 53BP1 (ab172580) 1:2000; GFP (A11122) 1:1000. Uncropped versions of immunoblots are provided in Supplementary Fig. 10.

Kovatcheva M., Liao W., Klein M.E., Robine N., Geiger H., Crago A.M., Dickson M.A., Tap W.D., Singer S, & Koff A. (2017). ATRX is a regulator of therapy induced senescence in human cells. Nature Communications, 8, 386.

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Immunofluorescence Analysis of Telomere-Associated Proteins

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Primary antibodies were TRF-2 (SantaCruz H-300, sc-9143, rabbit polyclonal, 20 µg/ml), γH2AX (Abcam, Anti-gamma H2A.X (phospho S139 antibody [9F3], ab26350, mouse monoclonal, 10 µg/ml), PML (PG-M3, SantaCruz, sc-966, mouse monoclonal, 2 µg/ml), anti-TRF1 (Abcam, TRF-78, ab10579), TIN2 (Abcam, ab197894), POT1 (Abcam, ab90552), RAP1 (Abcam, ab40144), ATRX (Abcam, ab97508), TopoIIIα (SantaCruz, sc13060, rabbit polyclonal), DAXX (Abcam, ab32140), LINE-1 (SantaCruz H110, sc67197, rabbit polyclonal), LINE-1 (chA1-L1-RNP, mouse monoclonal, 40 µg/ml) [46] , or β-Tubulin (9F3) rabbit IgG mAb (Cell Signaling, Cat. # 2128S, 1:10000, overnight, 4 °C), Anti-p21 antibody [EPR18021] (Abcam, ab188224), hTERT-antibody (Abcam, ab183105), Anti-Caspase-3 (Abcam, ab13847).

Aschacher T., Wolf B., Aschacher O., Enzmann F., Laszlo V., Messner B., Türkcan A., Weis S., Spiegl-Kreinecker S., Holzmann K., Laufer G., Ehrlich M, & Bergmann M. (2019). Long interspersed element-1 ribonucleoprotein particles protect telomeric ends in alternative lengthening of telomeres dependent cells. Neoplasia (New York, N.Y.), 22(2), 61-75.

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Indirect Immunofluorescence Assay for PML

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Our PML IF method was developed based on the indirect staining procedure previously described. 11, 15 Briefly, cytospin or smear preparations of peripheral blood or marrow cells were well air-dried and fixed in 1:1 methanol:acetone mixture at -20 o C for 90 s. Mouse monoclonal antihuman PML antibody (PG-M3; Santa Cruz Biotechnology, USA) was diluted 1:25 in phosphate buffered saline (PBS; containing 2% foetal calf serum and 0.01% sodium azide) and incubated with cells for 30 min at room temperature (RT). Slides were washed in a cold PBS bath for 5 min, then incubated at RT for 1 h with a secondary, sheep anti-mouse, fluorescein isothiocyanate (FITC) labelled immunoglobin G (Sigma-Aldrich, USA) diluted 1:100 as above. Slides were washed as above, mounted using Fluoprep (bioMerieux, France) and examined under an Olympus BH2 fluorescent microscope with a 40× oil lens and phase. Positive and negative controls of frozen samples (previous APL and other AML, respectively) were included in each testing round. Typical turn-around time of the test is 4 h. Results are phoned through to the requesting physician when the assay has been completed.

Chien N., Petrasich M., Chan G., Theakston E., Ruskova A., Eaddy N., Hawkins T., Berkahn L., Doocey R., Browett P.J., Green T.N, & Kalev-Zylinska M.L. (2019). Early treatment of acute promyelocytic leukaemia is accurately guided by the PML protein localisation pattern: real-life experience from a tertiary New Zealand centre. Pathology, 51(4).

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