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A2228

Manufactured by Merck Group
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The A2228 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of the A2228 is to perform specific tasks within a controlled laboratory environment.

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Lab products found in correlation

β actin, by Merck Group (8 mentions) Anti β actin, by Merck Group (6 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Nitrocellulose membrane, by Bio-Rad (5 mentions) F3165, by Merck Group (4 mentions) Image lab software, by Bio-Rad (4 mentions) Protease inhibitor cocktail, by Merck Group (4 mentions) Ripa buffer, by Merck Group (4 mentions) Odyssey infrared imaging system, by LI COR (3 mentions) Dc protein assay, by Bio-Rad (3 mentions) F1804, by Merck Group (3 mentions) Ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions) G8795, by Merck Group (3 mentions) N ethylmaleimide, by Merck Group (3 mentions) Mouse anti β actin, by Merck Group (3 mentions) Alexa fluor 488, by Thermo Fisher Scientific (3 mentions) Pierce ecl, by Thermo Fisher Scientific (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Bca kit, by Thermo Fisher Scientific (3 mentions) Ab133450, by Abcam (2 mentions)

145 protocols using a2228

1

Immunoblotting of EWS, CCDC6, and β-actin

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Monoclonal anti-EWS (Abcam, AB54708), monoclonal anti-CCDC6 (Abcam, AB56353), monoclonal anti-ß actin antibody (Sigma Aldrich, A2228) were used as primary antibodies at 1:3000, 1:1000 and 1:10000 dilutions respectively. Secondary goat anti-mouse IgG-HRP antibody (Santa Cruz biotechnology, sc-2005) was used at 1:10000 dilution.
Duggimpudi S., Larsson E., Nabhani S., Borkhardt A, & Hoell J.I. (2015). The Cell Cycle Regulator CCDC6 Is a Key Target of RNA-Binding Protein EWS. PLoS ONE, 10(3), e0119066.
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2

Detecting MMP20 in Cell Lysates and Media

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Concentrated media and cell lysates were run on the 11% SDS-polyacrylamide gel and subjected to Western blotting. Specifically, 50 ug cell lysate from each sample and 10 ul of concentrated media were used. After gel transfer to the PVDF membrane and blocking, MMP20 was detected by incubating the membrane with primary antibody overnight at 4°C and with secondary antibody for 2 h at room temperature. The primary antibodies used were a rabbit polyclonal anti-MMP20 antibody (ab39038, abcam plc., Cambridge, UK) and a mouse monoclonal anti-ACTB antibody (A2228, Sigma-Aldrich, St. Louis, MO, USA); both of which were diluted in 1:10,000.
Kim Y.J., Kang J., Seymen F., Koruyucu M., Gencay K., Shin T.J., Hyun H.K., Lee Z.H., Hu J.C., Simmer J.P, & Kim J.W. (2017). Analyses of MMP20 Missense Mutations in Two Families with Hypomaturation Amelogenesis Imperfecta. Frontiers in Physiology, 8, 229.
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3

Molecular Toolbox for Synaptic Protein Analysis

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SV2A and SV2B knockout mice were obtained from the Jackson Laboratory. Rat cDNAs encoding SV2A, SV2B, and SV2C were generously provided by R. Janz (Houston, TX). They were cloned into a lentiviral vector (Lox-Syn-Syn) as we previously described 7 (link). This vector contains two separate neuronal-specific synapsin promoters. One promoter drives expression of SV2 and the other one drives expression of GFP as a marker. Human cDNA encoding SV2C was obtained from PlasmID repository of Harvard Medical School. Human monoclonal antibody against HCA (RAZ-1) was generously provided by J. Marks (San Francisco, CA). Mouse monoclonal antibodies against VAMP2 (Cl 69.1), SNAP-25 (Cl 71.2), Syp (Cl 7.2) were generously provided by E. Chapman (Madison, WI) and are available from Synaptic Systems (Göttingen, Germany). SV2 (pan-SV2) was also provided by E. Chapman (Madison, WI) and it is available from The Developmental Studies Hybridoma Bank (AB2315387, Iowa City, IA). The following antibodies were purchased from indicated vendors: mouse monoclonal antibody against actin (Sigma, A2228); rabbit polyclonal antibody against synapsin (Millipore, Ab1543p); chicken polyclonal antibody against GFP (Aves lab, GFP-1020). Purified BoNT/A1 of Hall-A strain and BoNT/D of D1873 strain were generously provided by E. Johnson (Madison, WI).
Yao G., Zhang S., Mahrhold S., Lam K.H., Stern D., Bagramyan K., Perry K., Kalkum M., Rummel A., Dong M, & Jin R. (2016). N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A. Nature structural & molecular biology, 23(7), 656-662.
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4

Western Blot Analysis of REP1 Protein

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Cell samples or whole zebrafish larvae (10 per sample) were snap-frozen using dry ice. Samples were analyzed via Western blot assay, as previously described [21 (link)], using anti-REP1 (2F1 clone, Millipore #MABN52, RRID:AB_10808665, Burlington, MA, USA) primary antibody diluted 1:1000 followed by secondary anti-mouse IgG HRP conjugate diluted 1:10,000 (Sigma, St. Louis, MO, USA) in blocking solution (5% dry milk, PBS/0.1% Tween [PBS-T]). The membrane was stripped and re-probed with 1:5000 anti-β-actin antibody (Sigma-Aldrich #A2228, RRID:AB_476697, St. Louis, MO, USA) or anti-vinculin (Santa Cruz Biotechnology #sc-25336, RRID:AB_628438) as a loading control. Three independent experiments were performed to determine the mean protein expression in fibroblast samples.
Toualbi L., Toms M., Almeida P.V., Harbottle R, & Moosajee M. (2023). Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia. International Journal of Molecular Sciences, 24(20), 15225.
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5

Western Blot Analysis of ACE2, p38, and NF-κB

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Total proteins were extracted using RIPA buffer (Euromedex, Souffelweyersheim, France), and then equal amounts of proteins were reduced, size-separated on 12% stain-free precast SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA), and transferred to nitrocellulose membranes by using an iBlot2 apparatus (Thermo Fisher Scientific). The membranes were blocked in 5% milk in TBS-Tween 0.1% and incubated with specific primary antibodies overnight at 4°C; the antibodies were against ACE2 (AF933, R&D Systems, Minneapolis, MN, USA; 1:200), phospho- and total p38 (9211 and 9212, Cell Signaling Technology, Danvers, MA, USA; 1:2,000), phospho- and total NF-κB p65 (3039 and 8242, Cell Signaling Technology; 1:2,000), and β-actin (A2228, Sigma-Aldrich; 1:5,000). The blots were exposed to horseradish peroxidase-conjugated anti-rabbit (Cell Signaling Technology, 7074; 1:10,000) and anti-goat (A27104, Thermo Fisher Scientific; 1:2,000) secondary antibodies, and bound antibodies were detected using Clarity chemiluminescent substrate (Bio-Rad). Images were recorded using a Fujifilm LAS-3000 bioimaging system (Stamford, CT, USA).
Ruffin M., Bigot J., Calmel C., Mercier J., Givelet M., Oliva J., Pizzorno A., Rosa-Calatrava M., Corvol H., Balloy V., Terrier O, & Guillot L. (2021). Flagellin From Pseudomonas aeruginosa Modulates SARS-CoV-2 Infectivity in Cystic Fibrosis Airway Epithelial Cells by Increasing TMPRSS2 Expression. Frontiers in Immunology, 12, 714027.
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6

NF-κB Signaling Modulation in Chondrocytes and Synoviocytes

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The tuning of NF-κB signaling was further investigated by western blot analysis on both chondrocytes (2 × 105) and synoviocytes (6 × 104) plated in 24 well—plates in CTR (IL-1β stimulated) and sEV (IL-1β + sEV) conditions at both 4 and 15 h since sEV delivery. At the time of collection the medium was removed, the cells were recovered with a scraper using a small volume of cold PBS with the addition of inhibitors of phophatases and proteases. Then the cells were gently centrifuged and lysed with 20 µl of RIPA buffer. The samples were subsequently loaded, run and transferred to PVDF membranes as detailed previously.
Western blot was carried out with the following antibodies: Phospho-NF-κB p65(Ser536) (rabbit monoclonal antibody, clone 93H1, used at 1:1000, CELL SIGNALLING TECHNOLOGY #3033), and β-actin (mouse monoclonal, clone AC-74, used at 0.8 µg/ml SIGMA-ALDRICH # A2228) that served as loading control. Appropriate anti species HRP conjugated secondary antibodies were from JACKSON IMMUNORESEARCH.
Cavallo C., Merli G., Borzì R.M., Zini N., D’Adamo S., Guescini M., Grigolo B., Di Martino A., Santi S, & Filardo G. (2021). Small Extracellular Vesicles from adipose derived stromal cells significantly attenuate in vitro the NF-κB dependent inflammatory/catabolic environment of osteoarthritis. Scientific Reports, 11, 1053.
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7

Western Blot Analysis of SR-B1 in Liver

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Frozen liver samples were prepared for SDS–PAGE using a radioimmunoprecipitation assay buffer supplemented with protease inhibitors (Roche). Protein samples (15 μg per lane) were separated on a 10% Bis-Tris (pH 6.6) polyacrylamide gel using NuPAGE MES SDS Running Buffer under reducing conditions (Invitrogen). After the transfer to nitrocellulose membranes, blots were blocked for 2 h in PanReac Blocking buffer (AppliChem) and incubated for 1 h at room temperature with the primary antibody against SR-B1 (NB400-101; Novus Biologicals) and against β-actin (A2228; Sigma). Membranes were incubated for 90 min in secondary horseradish peroxidase conjugated antibodies (goat anti-rabbit, Jackson Immunoresearch, 1:5,000). Detection of protein bands was performed using a luminol/para-hydroxycoumarinic acid-based chemiluminescence substrate.
Bartelt A., John C., Schaltenberg N., Berbée J.F., Worthmann A., Cherradi M.L., Schlein C., Piepenburg J., Boon M.R., Rinninger F., Heine M., Toedter K., Niemeier A., Nilsson S.K., Fischer M., Wijers S.L., van Marken Lichtenbelt W., Scheja L., Rensen P.C, & Heeren J. (2017). Thermogenic adipocytes promote HDL turnover and reverse cholesterol transport. Nature Communications, 8, 15010.
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8

Western Blot Analysis of HIF-1 Signaling

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Cells were harvested, rinsed with PBS and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA,1 mM MgCl2, 0.5% NP-40, 1 mM Na3VO4, 1 mM NaF, protease inhibitors cocktail). Cell lysates were separated on SDS–polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories) and immunoblotted using the following primary antibodies. Rabbit anti-HIF-1α (1:1,000, GTX127309), anti-GST (1:5,000, GTX110736), anti-lamin B2 (1:5,000, GTX109894) and anti-tubulin (1:5,000, GTX112141) antibodies, as well as mouse monoclonal anti-HIF-1α antibody (1:1,000, GTX628480), were purchased from GeneTex. Mouse monoclonal antibodies recognizing β-actin (1:5,000, A2228) were purchased from Sigma. Mouse monoclonal HIF-1β antibody [2B10] (1:2,000, ab2771) were purchased from Abcam. Uncropped scans of the blots and gels are shown in Supplementary Fig. 19 in the Supplementary Information section.
Shih J.W., Chiang W.F., Wu A.T., Wu M.H., Wang L.Y., Yu Y.L., Hung Y.W., Wang W.C., Chu C.Y., Hung C.L., Changou C.A., Yen Y, & Kung H.J. (2017). Long noncoding RNA LncHIFCAR/MIR31HG is a HIF-1α co-activator driving oral cancer progression. Nature Communications, 8, 15874.
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9

Western Blot and 6mA Analysis

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For western blot analysis mouse α-FTO (ab92821, Abcam) and mouse α-ACTB (A2228, Sigma) were used. For 6mA-immunoprecipitation/miCLIP, rabbit α−6mA (202–003, Synaptic Systems) was used. For SmB-IP, a previously described mouse α-SmB antibody (clone 18F6)55 was used. For 6mA immunoblotting, a rabbit α−6mA antibody (ab190886, Abcam) was used.
Mauer J., Sindelar M., Despic V., Guez T., Hawley B.R., Vasseur J.J., Rentmeister A., Gross S.S., Pellizzoni L., Debart F., Goodarzi H, & Jaffrey S.R. (2019). FTO controls reversible m6Am RNA methylation during snRNA biogenesis. Nature chemical biology, 15(4), 340-347.
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10

Immunoblotting for ETV6 and Actin

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Total protein isolation was performed using RIPA-lysis buffer, supplemented with protease inhibitors and SDS-PAGE was executed according to standard protocols. Blotting was performed on a PVDF Membrane and the membrane blocked with 5% milk. For immunoblotting, the mouse monoclonal antibody against ETV6 (Abnova, H00002120-M01) was used in a 1:1000 dilution and the mouse monoclonal antibody against actin (Sigma, A2228) in a 1:5000 dilution. The anti-mouse secondary antibodies were used in a 1:5000 dilution.
Ghazavi F., Moerloose B.D., Loocke W.V., Wallaert A., Helsmoortel H.H., Ferster A., Bakkus M., Plat G., Delabesse E., Uyttebroeck A., Nieuwerburgh F.V., Deforce D., Roy N.V., Speleman F., Benoit Y., Lammens T, & Vlierberghe P.V. (2016). Unique long non-coding RNA expression signature in ETV6/RUNX1-driven B-cell precursor acute lymphoblastic leukemia. Oncotarget, 7(45), 73769-73780.
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