Rat anti mlkl

Manufactured by Merck Group

Sourced in United States

Rat anti-MLKL is a laboratory reagent used for the detection and analysis of MLKL (Mixed Lineage Kinase Domain-Like Protein) in research applications. MLKL is a protein that plays a key role in a form of programmed cell death called necroptosis. The Rat anti-MLKL antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to study the expression and localization of MLKL in biological samples.

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4 protocols using rat anti mlkl

Investigating Cell Signaling Pathways

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The following antibodies were used: rabbit anti-TXNIP (Cell Signaling, #14715), rabbit anti- PERK (Cell Signaling, #5683), rabbit anti-IL-18 (Abcam, ab68435), rabbit anti-NLRP3 (Abcam, ab214185), rabbit anti-IL-1β (Santa Cruz, sc-7884), rabbit anti-caspase-1 (Cell Signaling, #2225), mouse anti-Gasdermin D (Abcam, ab57785), mouse anti-β-actin (Cell Signaling), rabbit anti-BiP (Abcam, ab21685), rabbit anti-IRE1a (Abcam, ab48187), rabbit anti-phospho-MLKL (Ser358) (Cell Signaling, D6H3V), rat anti-MLKL (Millipore, MABC604), mouse anti-ASC (B-3) (Santa Cruz, sc-514414), rabbit anti-cleaved gasdermin D (Asp275) (Cell Signaling, #36425), goat anti-rabbit or mouse HRP-conjugated IgG (Cell Signaling), Alexa Fluor 488 donkey anti-rabbit or mouse IgG (Molecular Probes, A-21206), and Alexa Fluor 594 donkey anti-mouse IgG (Molecular Probes, A-21203). The following reagents were used: STF 083030 (IRE1a inhibitor, Santa Cruz, CAS 307543-71-1); resveratrol (TXNIP inhibitor, Sigma, R5010-100MG); PERK inhibitor II (Sigma, 5046510001); NLRP3 inhibitor (Sigma, 5381200001).

Cheng S.B., Nakashima A., Huber W.J., Davis S., Banerjee S., Huang Z., Saito S., Sadovsky Y, & Sharma S. (2019). Pyroptosis is a critical inflammatory pathway in the placenta from early onset preeclampsia and in human trophoblasts exposed to hypoxia and endoplasmic reticulum stressors. Cell Death & Disease, 10(12), 927.

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Immunohistochemical Identification of Glial and Necrotic Cells

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The slides were blocked with 0.01 M phosphate-buffered saline containing 3% bovine serum albumin and 0.3% Triton X-100 for 30 min, and were incubated with mouse anti-GFAP (1:500, Millipore, Billerica, MA, USA) for the identification of reactive astrocytes, rabbit anti-Iba-1 (1:1000, Wako, Osaka, Japan) for the identification of microglia/macrophages, rabbit anti-RIP3 (1:200, ENZO, New York, NY, USA) for the identification of necrotic cells or rat anti-MLKL (1:200, Millipore) for the identification of necrotic cells at room temperature overnight. After three washings in 0.01 M phosphate-buffered saline, the sections were incubated with the corresponding secondary antibodies for 4 h at room temperature in a dark environment. All sections were counterstained with Hoechst 33342 (Sigma, Santa Clara, CA, USA) to identify nuclei.

Fan H., Chen K., Duan L., Wang Y.Z, & Ju G. (2016). Beneficial effects of early hemostasis on spinal cord injury in the rat. Spinal Cord, 54(11), 924-932.

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Antibody Reagent Identification for Cell Analysis

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The rabbit anti-Bcl-X_L, the rabbit anti-caspase-3, the rabbit anti-LC3, the mouse anti-PARP1, the rabbit anti-actin antibodies were from Cell Signaling (ref. no. 2764, 9662, 2775, 9546, 4970 respectively). The rabbit anti-ATG6, the rabbit anti-total Bax and the rabbit anti-total Bak were from Santa Cruz Biotechnology (ref. no. sc-11427, sc-493 and sc-832 respectively). The mouse anti-caspase-1 was from Adipogen (ref. no. AG-20B-0048). The rabbit anti-ATG5 was from Abcam (ref. no. ab108327). The rat anti-MLKL was from Merck Millipore (ref. no. MABC604). The mouse FITC-labelled anti-CD19 was from Beckman Coulter (ref. no. A07768).

Heulot M., Chevalier N., Puyal J., Margue C., Michel S., Kreis S., Kulms D., Barras D., Nahimana A, & Widmann C. (2016). The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner. Oncotarget, 7(39), 64342-64359.

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Western Blot Analysis of Apoptosis Regulators

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Proteins were isolated from cells by resuspending in lysis buffer (described above). Lysates were clarified by centrifugation at 5000 g for 5 min at 4 C, and total protein concentration determined using Bradford reagent (Bio-Rad) and BSA standards. Equal quantities (20-50 mg) of total protein were separated under denaturing and reducing conditions (with 5% b-mercaptoethanol) using 4%-12% SDS-PAGE gels (Thermo Fisher). Proteins were transferred onto nitrocellulose membranes, blocked with 5% skim milk (Devondale) in PBS with 0.05% Tween-20 (PBST) for at least 1 h, and incubated overnight at 4 C with primary antibodies: rat anti-BAK (clone 4B5; WEHI in-house), rat anti-BAX (49F9; WEHI in-house), rat anti-caspase-8 (3B10; Enzo Life Sciences), rat anti-cIAP1 (1E1-1-10; Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck) and rabbit anti-b-actin-HRP (cat. #5125; Cell Signaling Technology). Membranes were washed with PBST and incubated for 1-2 h at room temperature with HRP-conjugated goat secondary antibodies (Southern Biotech). Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) was applied to membranes according to the manufacturer's instructions before imaging using a ChemiDoc Touch Imaging System (Bio-Rad) and analysis using Image Lab v6.0.0 software (Bio-Rad).

Stutz M.D., Allison C.C., Ojaimi S., Preston S.P., Doerflinger M., Arandjelovic P., Whitehead L., Bader S.M., Batey D., Asselin-Labat M.L., Herold M.J., Strasser A., West N.P, & Pellegrini M. (2021). Macrophage and neutrophil death programs differentially confer resistance to tuberculosis. Immunity, 54(8).

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