Whole spinal cord from Ipsilateral T13/L1 was sonicated in a mixture containing extraction buffer (Invitrogen) and protease inhibitors (Sigma). Ice-cold tissue samples were centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was removed, and the protein concentration for each sample was quantified using the Bradford method. Samples were heated to 75°C for 10 min and loaded into a standard polyacrylamide Bis-Tris gel (Invitrogen). SDS-PAGE was performed in 3-(N-morpholino)-propanesulfonic acid running buffer (Invitrogen) at 175 V for 1.25 h. Protein was transferred onto a nitrocellulose membrane using the iblot dry transfer system (Invitrogen). The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences) for 1 h and incubated with a primary antibody in blocking buffer overnight at 4°C. The following day, the membrane was washed in 1× PBS containing Tween 20 (0.1%) and then incubated in blocking buffer containing either goat anti-rabbit (NLRP3) or goat anti-mouse (B-actin) (LI-COR) IRDye 800CW secondary antibody at a concentration of 1:10,000 (LI-COR) for 1 h at room temperature. Protein expression was quantified using an Odyssey Infrared Imager (LI-COR) and expressed as a ratio to their housekeeping protein. Primary antibodies included rabbit anti-rat NLRP3 monoclonal antibody (1:1000; Abcam), and mouse anti-rat β-actin (1:200,000; Sigma-Aldrich).
Mouse anti rat β actin
Manufactured by Merck Group
Sourced in United States
Mouse anti-rat β-actin is a laboratory reagent used to detect the presence of the β-actin protein, a key structural component found in eukaryotic cells. It is a monoclonal antibody raised in mouse that specifically binds to the β-actin isoform expressed in rats.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imager, by LI COR (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Irdye 800cw secondary antibody, by LI COR (2 mentions)
Iblot dry transfer system, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mouse cxcr4, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Extraction buffer, by Thermo Fisher Scientific (1 mentions)
3 n morpholino propanesulfonic acid running buffer, by Thermo Fisher Scientific (1 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Rabbit anti rat nlrp3 monoclonal antibody, by Abcam (1 mentions)
Pvdf membrane, by Pall Corporation (1 mentions)
Phenylmethanesulfonyl fluoride, by Solarbio (1 mentions)
Mops running buffer, by Thermo Fisher Scientific (1 mentions)
Standard polyacrylamide bis tris gel, by Thermo Fisher Scientific (1 mentions)
5 protocols using mouse anti rat β actin
1
NLRP3 Protein Expression Analysis
2
Investigating CXCR4 and HIF-1α Expression
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BMSCs were cultured in the medium containing 1 µM ICA for 6, 12, 24, and 48 hours as previously described. The cells were then harvested and lysed in RIPA lysis buffer containing the protease inhibitor, phenylmethanesulfonyl fluoride (Solarbio Science & Technology Co., Ltd., Beijing, China), for 30 minutes at 4°C. Twenty micrograms of the sample was resolved by 10% SDS-PAGE and electro-transferred onto a PVDF membrane (Pall Corporation, Port Washington, NY, USA). The membranes were blocked and incubated with appropriate primary antibodies, including rabbit anti-rat CXCR4 and HIF-1α at dilutions of 1:400 and 1:1,000, respectively. To normalize protein loading, mouse anti-rat β-actin (Sigma-Aldrich Co., St Louis, MO, USA) antibody was used at a dilution of 1:5,000. After incubation with primary antibodies diluted in TBST with 5% nonfat milk at 4°C overnight, the membranes were exposed to the corresponding horseradish peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence. Protein band intensities on scanned films were compared with their respective controls using the ImageJ software (NIH, Bethesda, MD, USA). The bands were first rounded up using a volume Rect tool, and then target area intensity was calculated. The density of β-actin was used as control for the expression of CXCR4 and HIF-1α.
3
Western Blot Analysis of Protein Expression
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Samples were heated to 75°C for 10 min then loaded into a standard polyacrylamide Bis-Tris gel (Invitrogen). SDS-PAGE was performed in MOPS running buffer (Invitrogen) at 175 V for 75 min. Protein was transferred onto a nitrocellulose membrane using the iBlot dry transfer system (Invitrogen). The membrane was blocked with Odyssey blocking buffer (LI-COR) for 1 h and incubated with a primary antibody in blocking buffer overnight at 4°C. The following day, the membrane was washed in 1× PBS containing Tween 20 (0.1%) and then incubated in blocking buffer containing either goat anti-rabbit or goat anti-mouse (LI-COR) IRDye 800CW secondary antibody at a concentration of 1:10,000 for 1 h at room temperature. Primary antibodies included the following: mouse anti-rat high-mobility group box 1 (HMGB1) monoclonal antibody (1:4000; Abcam); rabbit anti-rat nod-like receptor protein 3 (NLRP3) monoclonal antibody (1:1000; Millipore); and mouse anti-rat β-actin (1:200,000; Sigma-Aldrich). Protein expression was quantified using an Odyssey Infrared Imager (LI-COR) and normalized to the housekeeping protein value for that sample, and data are presented as the percentage of the within-blot regular diet control samples.
4
Protein Extraction and Western Blotting
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Protein from skin tissues and cultured cells were extracted for Western blotting as described (Ai et al., 2007 (link)). Primary antibodies were rabbit anti-mouse KLF4 (1:1000, GenSpin,), rabbit anti-mouse CXCR4 (1:1000, eBioscience) and rat anti-mouse β-actin (1:1000, Sigma-Aldrich).
5
Protein Extraction and Western Blotting
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