For the total protein extraction, the treated cells were lysed by RIPA buffer (Beyotime, Shanghai, China) with protease inhibitors (Servicebio, Wuhan, China). Then, the same amount of whole-cell lysates was loaded on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. After 1 h of blocking, proteins were incubated with anti-β-actin antibodies (abs132001, Absin, Shanghai, China), anti-GAPDH antibodies (abs132004, Absin, Shanghai, China), and anti-BLM antibodies (abs122169, Absin, Shanghai, China) and subsequently with appropriate secondary antibodies (Servicebio Technology, Wuhan, China). Protein bands were detected and quantified via ImageLab (Bio-Rad, Hercules, CA, USA).
Abs132004
Manufactured by Absin
Sourced in China
The Abs132004 is a laboratory equipment designed for the precise measurement and analysis of various samples. It features advanced sensor technology and a compact, durable construction. The core function of this product is to provide accurate and reliable data for scientific research and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Abs20001, by Absin (2 mentions)
Abs20002, by Absin (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Abs132001, by Absin (1 mentions)
Protease inhibitor, by Wuhan Servicebio Technology (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Absin (1 mentions)
Ab184699, by Abcam (1 mentions)
Ab201015, by Abcam (1 mentions)
6 cm dish, by Jet Biofil (1 mentions)
Ab204448, by Absin (1 mentions)
Ripa lysis, by Ipsen (1 mentions)
Imager 600, by Merck Group (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Ab225844, by Abcam (1 mentions)
Ab172967, by Abcam (1 mentions)
Ab19645, by Abcam (1 mentions)
9 protocols using abs132004
1
Quantitative Protein Analysis Protocol
2
Protein Isolation and Western Blot Analysis
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Total protein was isolated using RIPA buffer and separated by 10% SDS-PAGE. After transferring to PVDF membranes, the blot was incubated with specific antibodies. Primary antibodies were as follows: GALNT2 (abs117502; Absin, Shanghai, China), ITGA5 (abs101364; Absin), ERK1 + ERK2 (ab184699; Abcam, Shanghai, China), p-ERK1 (T202) + p-ERK2 (T185) (ab201015; Abcam), Akt (ab8805; Abcam), p-Akt (T308) (ab38449; Abcam), and GAPDH (Absin; abs132004). Immunoreactive bands were visualized using an enhanced chemiluminescence kit (Absin).
3
Quantifying CRISPR-Cas9 Protein Expression
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Vectors expressing the editors (4 μg) and gRNA-puro (2 μg) were co-transfected into HEK293T cells in 6 cm dish (JETBIOFIL). Puromycin (InvivoGen) was added 24 h later to a final concentration of 2 μg/ml. Cells were harvested 72 h after transfection, and total protein extracted by RIPA lysis (EpiZyme). The protein samples were separated by SDS-PAGE, transferred to PVDF membrane (Merck Millipore). After blocking with 5% (w/v) non-fat milk dissolved in TBST (25 Mm Tris, PH 8.0, 150 Mm NaCl, and 0.1% Tween 20) for 1 h, the membranes were incubated overnight with anti-CRISPR-Cas9 antibody (Abcam # ab204448) or anti-GAPDH antibody (Absin #abs132004) at 4 °C. After extensive washing, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Proteins were visualized using Enhanced Chemiluminescence (ELC) reagent (Merck Millipore) and detected with an Amersham Imager 600.
4
Antibody-based Cardiac Protein Analysis
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The antibodies were used as follows: Anti-ANP antibody (ab225844, abcam, WB:1:1000), Anti-BNP antibody (ab19645, abcam, WB:1:500), Anti-β-MHC antibody (ab172967, abcam, WB:1:1000), Anti-Ythdf2 antibody (24744–1-AP, proteintech, IF:1:200; WB:1:1000), Anti-CPT-1a (66039-1-Ig, proteintech, IF:1:200, WB:1:1000), Anti-PPARα (AF5301, Affinity, WB:1:1000) GAPDH antibody (abs132004, absin, WB:1:5000), anti-β-tubulin antibody (10094-1-AP, Proteintech, WB:1:1000), goat anti-rabbit IgG-HRP (abs20002, absin, WB:1:10000), goat anti-mouse IgG-HRP (abs20001, absin, WB:1:10000). AngII (HY-13948) was purchased from MCE (China).
5
Quantifying SIRT1 Protein Levels
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Frozen placental tissue samples were homogenized in liquid nitrogen and lysed in cell lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein content was measured with the BCA kit (Beyotime Biotechnology, Shanghai, China). The Western blot analysis steps were conducted according to previously reported methods [38 (link)]. Primary antibodies against SIRT1(1:1000, 9475S, CST, Danvers, MA, USA) and GAPDH (1:1000, abs132004, Absin, Shanghai, China) were used in this study. Blots were analyzed with ImageJ software (NIH, Bethesda, MD, USA).
6
Western Blot Analysis of Cellular Proteins
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Protein was extracted from the cells using RIPA lysis buffer (CWBIO, China), followed by subjected to SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA for 1 hour at room temperature. Corresponding primary antibodies including anti-LIF (1:500, ab138002, Abcam), anti-STAT3 (1:1000, 9139, CST), anti-p-STAS3(Tyr705) (1:1000, 9145, CST), anti-CAV1 (1:1000, ab32577, Abcam), anti-SOX2 (1:1000, ab92494, Abcam), anti-ABCC2 (1:1000, ab172630, Abcam), anti-CDA (1:1000, ab222515, Abcam), anti-ZNRF1 (1:1000, ab175125, Abcam), anti-ubiquitin (1:1000, 3936, CST), anti-GAPDH (1:1,000, abs132004, Absin) were added to the membrane at 4℃ overnight. HRP-conjugated secondary antibodies were used. The protein bands were visualized by ECL detection system (Millipore, Germany).
7
Western Blot Analysis of TRPV1, TLR4, and MyD88
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SGD and spinal cord samples were centrifuged and immersed in RIPA buffer (P0013B, Beyotime, Shanghai, China) containing protease inhibitor (Thermo Fisher Scientific, Waltham, MA, United States) for the extraction of total protein content. The preparations were collected and stored at −80°C. The equal amounts of protein (10–50 μg) were separated by means of 10% SDS-PAGE, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were subsequently blocked with 5% skim milk for 2 h at 37°C, and incubated with primary antibodies against TRPV1 (ab6166, Abcam, Cambridge, UK), TLR4 (66350, abclonal, Wuhan, China) and MyD88 (ab219413, Abcam, Cambridge, UK) overnight at 4°C. The following day, immunoreactive bands were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (#7074 CST, MA, USA) for 30 minutes at room temperature. The ChemiDocXRS+ System (Bio-Rad, Hercules, CA) was utilized for imaging the bands, which were analyzed with the ImageJ software (NIH). The relative expression was calculated as the ratio of the intensity of the gene of interest to that of GAPDH (abs132004, absin, China) and β-Tubulin (#2148S CST, MA, USA).
8
Western Blot Analysis of Neurological Markers
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Equal amount of protein from the right hemisphere of the control or HI groups was separated on 4–12% SDS–PAGE gels (Beyotime) and transferred to polyvinyl difluoride membranes (Sigma Merck). The blots were then blocked with 5% bovine serum albumin in TBS buffer for 1 h at room temperature and probed overnight at 4°C by incubation with the following primary antibodies: Src (1:1,000, CST, catalog number: #2123), phosphor-Src (Tyr 416, 1:1,000, CST, catalog number: #6943), NMDAR subunit NR2B (1:1,000, CST, catalog number: #14544), phospho-NR2B (Tyr 1472, 1:1,000, CST, catalog number: #4208), myelin basic protein (MBP) (1:1,000, Biolegend, SMI99), Iba-1 (1:1,000, Abcam, ab178847), glial fibrillary acidic protein (GFAP) (1:1,000, CST, 12389), β-actin (1:5,000, Absin, abs137975), and GAPDH (1:1,000, Absin, abs132004), followed by appropriate secondary HRP-conjugated antibodies (anti-rabbit, 1:5,000, Absin, abs20002; anti-mouse, 1:5,000, Absin, abs20001). Blots were visualized with enhanced chemiluminescence reagents (BD Pharmingen) and Bio-Rad Image software.
9
Western Blot Analysis of BRD4, NLRP3, and SIRT1
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The expression levels of BRD4, NLRP3, and SIRT1 in heart tissue and H9C2 cells were determined by Western Blotting. The protein samples were electrophoretic with 10% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) and then transferred to polyvinylidene uoride (PVDF) membranes at 200 mA constant current. Then, after sealing with 5% skim milk powder at room temperature for 1 h, the anti-BRD4(1:1000;Abcam,ab128874),anti-SIRT1(1:1000;Abcam,ab189494), anti-NLRP3(1:1000;Cell Signaling Technology,15101S), anti-Caspase-1(1:1000; Cell Signaling Technology,3866T), anti-Cleaved caspase-1(1:1000;Cell Signaling Technology,4199T),anti-GSDMD(1:1000;Abclonal, A18281), and anti-GAPDH(1:1000,Absin,abs132004)were incubated at 4℃overnight.The membrane was washed three times and then incubated with secondary antibody at room temperature for 1 h. The protein bands were revealed by ECL (Beyotime Institute of Biotechnology, China) chemiluminescence solution. The Image J Image analysis system (NIH, USA) was used to quantify protein expression.GAPDH is used as an internal reference for Western Blotting.
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