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Mabc604

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MABC604 is a laboratory equipment used for sample preparation and analysis. It is designed to perform automated mixing and blending of various materials and solutions. The core function of MABC604 is to ensure consistent and reproducible sample homogenization, which is essential for accurate and reliable analytical results.

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Lab products found in correlation

Ab187091, by Abcam (4 mentions) Ab53444, by Abcam (2 mentions) Nbp1 77299, by Merck Group (2 mentions) Fluoromount g, by Southern Biotech (2 mentions) Anti actin, by Merck Group (2 mentions) Anti gapdh, by Cell Signaling Technology (2 mentions) Sc 47778 hrp, by Santa Cruz Biotechnology (2 mentions) Mabc1640, by Merck Group (2 mentions) Ab10527, by Merck Group (1 mentions) Anti ripk1, by BD (1 mentions) Anti vdac, by Merck Group (1 mentions) Nvp bep800, by Selleck Chemicals (1 mentions) Anti hsp90 adi spa 835, by Enzo Life Sciences (1 mentions) Q vd oph, by R&D Systems (1 mentions) 17 aag, by Selleck Chemicals (1 mentions) Mouse anti flag m2, by Merck Group (1 mentions) Mouse anti actin a1978, by Merck Group (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Luminol reagent, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions)

Spelling variants of this product

MLKL (MABC604) (2 citations)

8 protocols using mabc604

1

Immunohistochemical Analysis of Cuprizone-Induced Demyelination

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Paraffin-embedded sections of tissue from cuprizone-fed mice underwent
deparaffinization using Histoclear (2 x 10 min) and a gradient of ethanol (EtOH)
concentrations, each for 5 minutes in the following order: 2 x 100%, 1 x 95%, 1
x 70% and 1 x 50%, before being washed in Tris-buffered saline (TBS) for 3 x 5
minute washes. Slides were then placed in Vector Unmasking Solution under high
heat and pressure for 20 minutes before a final TBS wash. Slides were
permeabilized and blocked for 1 hour (5% horse serum and 0.3% Triton-X-100 in
PBS) before incubation with primary antibodies overnight at 4°C in a
humid chamber. Microglia/macrophages were detected with rat anti-CD68 (Abcam,
Ab53444, 1: 100) and cell death was assessed using rabbit anti-RIPK3 (Novus
Biologicals, NBP1-77299, 1:100) and rat anti-MLKL (Merck-Millipore, MABC604,
1:100). Following washes in PBS, fluorescently conjugated secondary antibodies
were applied (Invitrogen, 1:1000). Slides were counterstained with Hoechst and
coverslipped with Fluoromount-G (Southern Biotech). Images were acquired with an
Olympus spinning disk confocal microscope using a 60X objective with Slidebook 6
software. 3 animals were analyzed per time point.
Lloyd A.F., Davies C.L., Holloway R.K., Labrak Y., Ireland G., Carradori D., Dillenburg A., Borger E., Soong D., Richardson J.C., Kuhlmann T., Williams A., Pollard J.W., des Rieux A., Priller J, & Miron V.E. (2019). Central nervous system regeneration is driven by microglia necroptosis and repopulation. Nature neuroscience, 22(7), 1046-1052.
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2

Antibody-based detection of MLKL, GAPDH, and Actin

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Primary antibodies used in this study were: rat anti-MLKL (clone 3H1, produced in-house1 (link); available as MABC604, EMD Millipore, Billerica, MA, USA; 1:1000), rabbit anti-MLKL phospho-S358 (AB187091, Abcam; 1:4000), anti-GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA; 1:2000), anti-Actin (A-1987, Sigma-Aldrich, St Louis, MO, USA; 1:3000), and anti-VDAC1 (AB10527, EMD Millipore; 1:5000). Recombinant hTNF-Fc, produced in-house, and the Smac mimetic, Compound A, have been previously described38 (link),39 (link). The pan-caspase inhibitor, IDN-6556/emricasan, was provided by Tetralogic Pharmaceuticals.
Petrie E.J., Sandow J.J., Jacobsen A.V., Smith B.J., Griffin M.D., Lucet I.S., Dai W., Young S.N., Tanzer M.C., Wardak A., Liang L.Y., Cowan A.D., Hildebrand J.M., Kersten W.J., Lessene G., Silke J., Czabotar P.E., Webb A.I, & Murphy J.M. (2018). Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis. Nature Communications, 9, 2422.
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3

Western Blot Analysis of Cell Lines

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The following primary antibodies were used for western blots of both mouse and human cell lines: anti-MLKL (produced in-house;25 (link) available as MABC604, EMD Millipore, Billerica, MA, USA), anti-GAPDH (2118, Cell Signalling Technology, Danvers, MA, USA), anti-Actin (A-1987, Sigma-Aldrich, St Louis, MO, USA), anti-RIPK1 (610458, BD Biosciences, Franklin, NJ, USA), anti-Hsp90 (ADI-SPA-835, Enzo, Life Sciences, Farmingdale, NY, USA), anti-Cdc37 (D11A3, Cell Signalling Technology), and anti-VDAC (Sigma-Aldrich). Anti-mouse RIPK3 (PSC-2283-c100, Axxora, San Diego, CA, USA) and anti-human RIPK3 (ab56164, Abcam, Cambridge, UK) were used for their respective cell lines.
Recombinant hTNF-Fc, produced in-house, and the Smac mimetic, Compound A, were described previously.71 (link), 72 (link) QVD-OPh was obtained from R&D Systems (Minneapolis, MN, USA). Both NVP-BEP800 and 17-AAG were obtained from Selleckchem (Sydney, NSW, Australia), and AT13387 was produced by Active Biochem (Wanchai, Hong Kong).
Jacobsen A.V., Lowes K.N., Tanzer M.C., Lucet I.S., Hildebrand J.M., Petrie E.J., van Delft M.F., Liu Z., Conos S.A., Zhang J.G., Huang D.C., Silke J., Lessene G, & Murphy J.M. (2016). HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death. Cell Death & Disease, 7(1), e2051-.
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4

Immunohistochemical Analysis of Cuprizone-Induced Demyelination

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Paraffin-embedded sections of tissue from cuprizone-fed mice underwent
deparaffinization using Histoclear (2 x 10 min) and a gradient of ethanol (EtOH)
concentrations, each for 5 minutes in the following order: 2 x 100%, 1 x 95%, 1
x 70% and 1 x 50%, before being washed in Tris-buffered saline (TBS) for 3 x 5
minute washes. Slides were then placed in Vector Unmasking Solution under high
heat and pressure for 20 minutes before a final TBS wash. Slides were
permeabilized and blocked for 1 hour (5% horse serum and 0.3% Triton-X-100 in
PBS) before incubation with primary antibodies overnight at 4°C in a
humid chamber. Microglia/macrophages were detected with rat anti-CD68 (Abcam,
Ab53444, 1: 100) and cell death was assessed using rabbit anti-RIPK3 (Novus
Biologicals, NBP1-77299, 1:100) and rat anti-MLKL (Merck-Millipore, MABC604,
1:100). Following washes in PBS, fluorescently conjugated secondary antibodies
were applied (Invitrogen, 1:1000). Slides were counterstained with Hoechst and
coverslipped with Fluoromount-G (Southern Biotech). Images were acquired with an
Olympus spinning disk confocal microscope using a 60X objective with Slidebook 6
software. 3 animals were analyzed per time point.
Lloyd A.F., Davies C.L., Holloway R.K., Labrak Y., Ireland G., Carradori D., Dillenburg A., Borger E., Soong D., Richardson J.C., Kuhlmann T., Williams A., Pollard J.W., des Rieux A., Priller J, & Miron V.E. (2019). Central nervous system regeneration is driven by microglia necroptosis and repopulation. Nature neuroscience, 22(7), 1046-1052.
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5

Antibodies for MLKL and RIPK3 Studies

Cited 1 time
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Primary antibodies used in this study were: rat anti-MLKL (clone 3H1, produced in-house; 1:1000 dilution; available as MABC604, EMD Millipore, Billerica, MA, USA), rat anti-human MLKL pseudokinase domain (clone 7G2, produced in-house; 1:2000 dilution)32 (link), rabbit anti-human MLKL phospho-S358 (AB187091, Abcam; 1:3000), mouse anti-Actin (A-1978, Sigma-Aldrich, St Louis, MO, USA; 1:5000), rat anti-human RIPK3 (clone 1H2, produced in-house; 1:1000)17 (link), rabbit anti-human phospho-S227 (D6W2T, CST, 1:1000), mouse anti-FLAG M2 (Sigma-Aldrich, F1804, 1:3000). Recombinant hTNF-Fc, produced in-house, and the Smac mimetic, Compound A, have been previously described63 (link),64 (link). The pan-caspase inhibitor, IDN-6556/emricasan, was provided by Tetralogic Pharmaceuticals.
Garnish S.E., Meng Y., Koide A., Sandow J.J., Denbaum E., Jacobsen A.V., Yeung W., Samson A.L., Horne C.R., Fitzgibbon C., Young S.N., Smith P.P., Webb A.I., Petrie E.J., Hildebrand J.M., Kannan N., Czabotar P.E., Koide S, & Murphy J.M. (2021). Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis. Nature Communications, 12, 2211.
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6

Quantitative Western Blot Analysis of Cell Death Signaling

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Snap-frozen brain tissue was homogenized with ice-cold RIPA lysis buffer and the supernatant was collected as previously described [20 (link)]. 20 μg of total protein lysate was used for SDS-PAGE and transferred onto PVDF membranes (Bio-Rad, CA, USA). The membranes were incubated with mouse monoclonal anti-RIPK1 (1:1000, 610458, BD Pharmingen, CA, USA), rabbit monoclonal anti-phospho-RIPK1 (Ser166) (1:1000, 65746S, Cell Signaling), rabbit polyclonal anti-RIPK3 (1:1000, ab56164, Abcam), rat monoclonal anti-MLKL (1:1000, MABC604, Merck Millipore, MA, USA), rabbit monoclonal anti-phospho-MLKL (Ser345) (1:1000, 37333S, Cell Signaling), mouse monoclonal anti-IL-1β (1:1000, MCA1397, AbD Serotec, Kidlington, UK), rabbit polyclonal anti-H3 (1:2000, 9715S, Cell Signaling) or mouse monoclonal anti-GAPDH (1:10000, KC-5G4, KANGCHEN, Shanghai, China) antibody overnight at 4 ºC, and then incubated with anti-mouse/rabbit IgG peroxidase-conjugated secondary antibody (Santa Cruz, CA, USA). Signal was visualized with Western blotting Luminol Reagent (Santa Cruz), detected using ChemiDoc™ Touch Imaging System (Bio-Rad) and quantified with Image J software (V1.48, National Institutes of Health, USA). Beta-actin (1:10000, A5441, Sigma) was used for signal normalization.
Deng X.X., Li S.S, & Sun F.Y. (2019). Necrostatin-1 Prevents Necroptosis in Brains after Ischemic Stroke via Inhibition of RIPK1-Mediated RIPK3/MLKL Signaling. Aging and Disease, 10(4), 807-817.
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7

Antibodies and Reagents for MLKL and RIPK3

Cited 1 time
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Primary antibodies used in this study were: rat anti-MLKL (clone 3H1, produced in-house; 1:1000 dilution; available as MABC604, EMD Millipore, Billerica, MA, USA), rat anti-human MLKL pseudokinase domain (clone 7G2, produced in-house32 (link); 1:2000 dilution; soon available from EMD Millipore as MABC1636), rabbit anti-human MLKL phospho-S358 (AB187091, Abcam; 1:2000), mouse anti-Actin (C4) HRP (sc-47778 HRP, Santa Cruz Biotechnology; 1:10000), rat anti-human RIPK3 (clone 1H2, produced in-house11 (link); 1:1000; available as MABC1640, EMD Millipore, Billerica, MA, USA), rabbit anti-human RIPK3 phospho-S227 (D6W2T, CST; 1:2000). Recombinant hTNF-Fc62 (link) was produced in-house, while the Smac mimetic, Compound A63 (link) and the pan-caspase inhibitor, IDN-6556/Emricasan, were provided by Tetralogic Pharmaceuticals. The RIPK3 inhibitors, GSK′843 (ref. 54 (link)) and BMS Compound 10 (ref. 48 (link)), were kindly provided by Anaxis Pty Ltd (Australia).
Meng Y., Davies K.A., Fitzgibbon C., Young S.N., Garnish S.E., Horne C.R., Luo C., Garnier J.M., Liang L.Y., Cowan A.D., Samson A.L., Lessene G., Sandow J.J., Czabotar P.E, & Murphy J.M. (2021). Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis. Nature Communications, 12, 6783.
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8

Immunoblotting of Necroptosis Mediators

Cited 1 time
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Primary antibodies used in this study were: rat anti-human RIPK3 (clone 1H2, produced in-house; 1:1000; available as MABC1640, EMD Millipore, Billerica, MA, USA) [25 (link)], rat anti-mouse RIPK3 (clone 1H12, produced in-house; 1:1000) [41 (link)], rabbit anti-human RIPK3 phospho-S227 (D6W2T, CST, 1:2000), rabbit anti-human RIPK3 phospho-S227 (EPR9627, Abcam; 1:2000) rat anti-human MLKL pseudokinase domain (clone 7G2, produced in-house; 1:2000 dilution; available as MABC1636, EMD Millipore, Billerica, MA, USA) [27 (link)], rat anti-human MLKL (3H1, produced in-house; 1:1000; available as MABC604, EMD Millipore, Billerica, MA, USA) [25 (link)], mouse anti-human RIPK1 (610459, BD Transduction Laboratories; 1:1000), rabbit anti-human MLKL phospho-S358 (AB187091, Abcam; 1:2000), mouse anti-GAPDH (MAB374, Millipore; 1:2000) and anti-Actin (C4) HRP (sc-47778 HRP, Santa Cruz Biotechnology; 1:10,000). The Smac mimetic, Compound A [42 (link)] and the pan-caspase inhibitor, IDN-6556/Emricasan, were provided by Tetralogic Pharmaceuticals, and recombinant hTNF-Fc [43 (link)] was produced in-house.
Meng Y., Horne C.R., Samson A.L., Dagley L.F., Young S.N., Sandow J.J., Czabotar P.E, & Murphy J.M. (2022). Human RIPK3 C-lobe phosphorylation is essential for necroptotic signaling. Cell Death & Disease, 13(6), 565.
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