Hippocampus tissues were harvested at ages P14, 21, 28, 35, and 42 and then homogenized using RIPA lysis buffer (CW2333s, CW, biotech), supplemented with 0.1 mM phenylmethylsulphonyl fluoride (PMSF)‐protease inhibitors (CW2200S, CW, biotech). Protein concentration was determined by a BCA protein assay kit (GK10009, GLPBIO). Equal amounts of protein samples (20 μg) were loaded and separated by 10% or 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (G2037‐50T, Servicebio) transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 3% non‐fat milk for 1 h at room temperature, membranes were incubated at 4°C overnight with primary antibodies of chicken anti‐SynCAM1(1:1000, CM004‐3, MBL), rabbit anti‐PV (1:500, A13538, ABclonal), or rat anti‐β‐actin (1:10000, 660009‐1, Proteintech). After washing with Tris Buffer Saline with Tween‐20 (TBST), the membranes were incubated for 2 h at room temperature with secondary antibodies of goat anti‐rabbit IgG (1:10,000, SA00001‐2, Proteintech), goat anti‐mouse IgG (1:10,000, SA00001‐2, Proteintech), and goat anti‐chicken IgG (1:5000, L35001, Signalway) and visualized with an enhanced chemiluminescent detection (ECL) kit (WBKLS0100, Millipore) and analyzed with Image J software.
Sa00001 2
Manufactured by Proteintech
Sourced in United States, China, United Kingdom
The SA00001-2 is a laboratory equipment product. It serves as a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Sa00001 1, by Proteintech (206 mentions)
Pvdf membrane, by Merck Group (48 mentions)
Ripa lysis buffer, by Beyotime (25 mentions)
Bca protein assay kit, by Beyotime (22 mentions)
Ripa buffer, by Beyotime (15 mentions)
Gapdh, by Proteintech (12 mentions)
Amersham imager, by Cytiva (12 mentions)
Ipvh00010, by Merck Group (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Ab32503, by Abcam (8 mentions)
Ab8245, by Abcam (8 mentions)
Ripa buffer, by Solarbio (8 mentions)
β actin, by Proteintech (7 mentions)
Polyvinylidene difluoride membrane, by Merck Group (7 mentions)
Bca kit, by Beyotime (7 mentions)
Quantity one software, by Bio-Rad (7 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Goat anti rabbit igg, by Proteintech (6 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Ab5694, by Abcam (5 mentions)
405 protocols using sa00001 2
1
Hippocampal SynCAM1 and PV Expression
2
Quantifying Protein Expression in Lung Tissues
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The lung tissues/cells were homogenized in RIPA buffer containing protease and phosphatase inhibitors. The lysates were centrifuged at 14,000 rpm (15 min, 4 °C), and the supernatant was collected for further analysis. The proteins were separated with SDS-polyacrylamide gels and then transferred to a polyvinyl difluoride (PVDF) membrane (Millipore, IPVH00010). Immunoblotting was performed at 4 °C overnight using primary antibodies directed against FASN (1:2000, Abcam, ab22759), p38 MAPK (1:500, Proteintech, 14064-1-AP), phospho (p)-p38 MAPK (Thr180/Tyr182) (1:1000, Proteintech, 28796-1-AP), NLRP3 (1:500, wanleibio, WL02635), VE-cadherin (1:1000, Affinity, AF6265), Drp1 (1:2000, Proteintech, 12957-1-AP), phospho (p)-Drp1 (Ser616) (1:500, Affinity, AF8470), Mfn2 (1:1000, abclonal, A19678) and β-actin (1:2000, Proteintech, 60,008–1-Ig). The membranes were then incubated with the secondary antibody (1:10,000, SA00001-1, SA00001-2, Proteintech) at room temperature for 1 h. The protein bands were detected using ECL solution (E003, 7 Sea Biotech, China) and visualized using the ImageJ software (v.1.53c; NIH).
3
Comprehensive Western Blot Analysis
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The western blot analysis procedure was conducted as previously reported11. The primary or secondary antibodies and their respective diluted concentrations we used in this study are as follows: (LHPP: dilution 1:200, catalog no. 15759‑1‑AP, Proteintech; GAPDH, dilution 1:2000, catalog no. 60004‑1‑AP, Proteintech; β-actin, dilution 1:2000, catalog no. 20536‑1‑AP, Proteintech; AKT, dilution 1:500, catalog no. 10176‑2‑AP, Proteintech; p‑AKT, dilution 1:1000, catalog no. 4060S, CST; PI3K, dilution 1:1000, catalog no. 4249S, CST; p-PI3K, dilution 1:1000, catalog no. ab278545, abcam; E-cadherin, dilution 1:1000, catalog no. 9782T, CST; β-catenin, dilution 1:1000, catalog no. 9782T, CST; N-cadherin, dilution 1:1000, catalog no. 9782T, CST; Vimentin, dilution 1:1000, catalog no. 9782T, CST; Snail , dilution 1:1000, catalog no. 9782T, CST; Slug, dilution 1:1000, catalog no. 9782T, CST; ZEB1, dilution 1:1000, catalog no. 9782T, CST; mTOR, dilution 1:1000, catalog no. 9862T, CST; p-mTOR, dilution 1:1000, catalog no. 9862T, CST; p-pS6k(C371), dilution 1:1000, catalog no. 9862T, CST; p-pS6k(T389), dilution 1:1000, catalog no. 9862T, CST; 1-Histidine phosphorylation (1- PHis), dilution 1:1000, catalog no. MABS1330, Merk; 3-PHis, dilution 1:1000, catalog no. MABS1352, Merk); secondary antibody (dilution 1:10000, catalog no. SA00001-2/ SA00001-1, Proteintech).
4
Quantitative Western Blot Analysis
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The bronchial epithelial cells were treated with 200 μl RIPA for 10 min on ice, and then were centrifuged at 12,000×g (4 °C) for 15 min. The loaded proteins (50–170 μg) were separated on a 10% SDS-PAGE, followed by transferring onto PVDF membranes. The samples were blocked with TBS-Tween 20 (TBST) containing 5% skim milk for 60 min at room temperature, and at 4 °C overnight, then 30 min at room temperature. The membranes were incubated with rabbit anti-mouse antibody against mouse C-EBPβ (1:1000, ab53138; Abcam, Cambridge, UK), and mouse anti-mouse antibody against β-actin (1:5000, 60008-1-Ig; Proteintech, IL, USA) for 90 min at room temperature, and then they were incubated with horseradish peroxidase conjugated goat anti-rabbit (1:6000, SA00001-2; Proteintech, IL, USA) or anti-mouse antibody (1:5000, SA00001-1; Proteintech, IL, USA) for 90 min at room temperature. At last, blots were developed with the ECL Plus reagents (Thermo pierce, IL, USA).
5
Characterization of GBM-derived EVs and M2 Macrophage Lysates
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Human GBM cell U87MG‐derived EVs and cell lysates from M2 macrophages were collected in RIPA lysis buffer (Beyotime, China). The protein concentrations of purified EVs and total protein extracted from M2 cells were assessed using the bicinchoninic acid (BCA) protein assay kit (Vazyme, China). The cell lysates (40 μg) and sEVs proteins (5 μg) were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Merck Millipore, USA). Membranes were blocked and incubated with primary antibodies at 4°C overnight. β‐tubulin and GAPDH were used for loading controls. After incubation of horseradish peroxidase (HRP)‐conjugated anti‐mouse or anti‐rabbit secondary antibodies (SA00001‐1 and SA00001‐2; 1:5000; Proteintech, China) at room temperature (RT) for 1 h, blots were visualized using ECL Plus western blotting detection system.
6
Immunohistochemical Analysis of ccRCC
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IHC was performed as previously described 39 (link). Firstly, ccRCC samples were fixed in formalin, dehydrated, and embedded in paraffin. Secondly, sample sections were incubated with corresponding primary antibodies overnight at 4 °C. Thirdly, sample sections were washed with PBS three times. Finally, sample sections were incubated with corresponding secondary antibodies at room temperature for 2h. Moreover, ImageJ software was used to calculate the mean OD value of cells with positive staining.
The primary antibodies and secondary antibodies were as following: MMP9 (1:100, Abclonal, China, A0289); IGFBP1 (1:150, Abclonal, China, A11109); CD14 (1:200, Proteintech, China, 60253-1-lg); secondary antibodies (1:3000, Proteintech, China, SA00001-1 and SA00001-2).
The primary antibodies and secondary antibodies were as following: MMP9 (1:100, Abclonal, China, A0289); IGFBP1 (1:150, Abclonal, China, A11109); CD14 (1:200, Proteintech, China, 60253-1-lg); secondary antibodies (1:3000, Proteintech, China, SA00001-1 and SA00001-2).
7
Western Blot Analysis of SARS-CoV-2 and Host Factors
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Cells from one confluent well of a six-well plate were lysed in radioimmunoprecipitation assay lysis buffer (1×) supplemented with protease inhibitor cocktail (Cell Signaling Technology). Total cell lysate (15 to 20 μg) was separated by 4 to 20% bis-tris SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore). Then, the membranes were blocked in 5% nonfat dry milk and inoculated with the following antibodies overnight: anti-human AhR (catalog no. GTX129013, GeneTex), anti–SARS-CoV-2 NP (catalog no. MA5-36270, Invitrogen), anti–glyceraldehyde-3-phosphate dehydrogenase (catalog no. ab181602, Abcam), and anti-ACE2 (catalog no. ab108252, Abcam). Membranes washed four times with tris-buffered saline and 0.1% Tween 20 were incubated with horseradish peroxidase–conjugated secondary antibodies (goat anti-rabbit; catalog no. SA00001–2, Proteintech; goat anti-mouse; catalog no. SA00001-1, Proteintech) and developed using enhanced chemiluminescence substrate. The results were confirmed by at least three biological replicates.
8
Protein Expression Analysis in ESCs
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Protein extraction was prepared from ESCs using RIPA buffer (Beyotime Biotechnology) with proteinase and phosphatase inhibitors. After being quantified by the BCA assay kit (Thermo Scientific), equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (Merck Millipore). Membranes were blocked in 5% BSA (Sigma) at room temperature for 1 h, followed by incubation with primary antibodies against GAPDH (1:10000; Proteintech, 60004-1-Ig), GLS1 (1:1000; Abcam, ab93434), Collagen I (1:1000; Abcam, ab34710), α-SMA (1:1000; Abcam, ab7817), MFN1(1:1000; Abcam, ab57602), MFN2(1:1000; Abcam, ab56889), DRP1(1:1000; Abcam, ab56788), PINK1(1:1000; Cell Signaling Technology, 6946), p-p70-S6K (1:1000; Cell Signaling Technology, 9234), p-4E-BP1 (1:1000; Cell Signaling Technology, 2855) and p-S6 (1:1000; Cell Signaling Technology, 4858) overnight at 4 °C. The membrane was then incubated with HRP-conjugated goat anti-rabbit (1:10000; Proteintech, SA00001-2) or goat anti-mouse (1:10000, Proteintech, SA00001-1) secondary antibodies at room temperature for 1 h. Pageruler (ThermoFisher, 26,616) was used as the molecular marker. Protein expression was visualized with an ECL detection kit (Sigma-Aldrich). GAPDH was used as the loading control.
9
Liver Protein Extraction and Western Blot Analysis
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The liver was weighed and ground with liquid nitrogen. The milled tissue powder was added into lysis solution containing RIPA (tissue weight: volume of lysis solution 20 mg:150–250 μL) for lysis. BCA protein detection kit was used to detect protein concentration. The total protein (10 μg) of each sample was added to SDS-PAGE gel electrophoresis to separate the protein and transferred to PVDF membrane (Millipore). The PVDF membrane containing protein was incubated in a closed solution for 2 hours and then incubated in the required primary antibody, including TLR4 antibody (1 : 500, ab13556; Abcam), MyD88 antibody (1 : 1000, ab219413; Abcam), TRAF6 (1 : 1000, ab33915; Abcam), IκB antibody (1 : 2000, ab76429; Abcam), P-IκB antibody (1 : 1000, 2859 S; Abcam), NF-κB antibody (1 : 2000, ab16502; Abcam), and β-actin antibody (1 : 10000, 20536-1-AP; Proteintech), incubated at 4°C overnight. After incubating with HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1 : 5000, SA00001-2, Proteintech) and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (1 : 5000, SA00002-1, Proteintech) for 1 h, the PVDF membrane was washed with solution TBST, treated with chemiluminescence reagent, taken photos by exposure, and performed quantitative analysis with Image-Pro Plus 6.0.
10
Western Blot Analysis of VSMC Markers
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VSMCs in each group were collected and lysed in 100 μL of RIPA lysis buffer (Beyotime, Shanghai, China). Then, the total protein was extracted and the protein concentration was determined by Bradford method. The equivalent amount of protein in each group was separated by SDS-PAGE and then transferred onto PVDF membrane (Millipore, Bedford, MA, USA). Then, the membrane was blocked in 5% skim milk at room temperature for 1 h before being incubated with the primary antibody overnight at 4°C. Next, secondary antibody was added and the membranes were incubated at room temperature for 1 h. Electrochemiluminescence substrate A and B solutions (Millipore, Bedford, MA, USA) and Amersham Imager 600 (GE Healthcare, Chicago, IL, USA) were used to visualize the protein bands. The ImageJ software (NIH, Bethesda, Maryland, USA) was utilized for quantifying the protein bands. The antibodies used in this study included anti-PIK3CG (1:1000, 140,307, Abcam, Cambridge, UK), anti-SM-22α (1:1000, ab14106, Abcam, Cambridge, UK), anti-α-SMA (1:1000, ab184705, Abcam, Cambridge, UK), anti-smooth muscle myosin heavy chain 11 (SMMHC) (1:1000, ab133567, Abcam, Cambridge, UK), anti-Calponin (1:1000, ab227661, Abcam, Cambridge, UK), anti-GAPDH (1:3000, 60,004-1-Ig, Proteintech, Wuhan, China), and the secondary antibody (1:2000, SA00001-2, Proteintech, Wuhan, China).
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