Total proteins were extracted by RIPA. Proteins were separated by SDS-PAGE and electro-transferred onto PVDF membranes. The membranes were soaked in buffer containing 5% BSA for 1 h and then incubated with primary antibodies including anti‐p-mTOR (5536s, 1:1000, Cell Signaling Technology), anti‐mTOR (2983s, 1:1000, Cell Signaling Technology), anti‐p-PI3K (bs-6417R, 1:1000, Bioss), anti‐PI3K (bs-10657R, 1:1000, Bioss), anti‐p-AMPK (2531S, 1:1000, Cell Signaling Technology), anti‐AMPK (5832S, 1:1000, Cell Signaling Technology), anti‐p-AKT (Sc-293125, 1:1000, Santa), anti‐AKT (Sc-5298, 1:1000, Santa), anti‐PGC1α (bs-7535R, 1:1000, Bioss), anti‐LC3B (ab51520, 1:5000, Abcam), anti‐ZO-1 (61–7300, 1:1000, Invitrogen), anti‐Occludin (71–1500, 1:1000, Invitrogen), or anti‐Claudin-5 (34–1600, 1:1000, Invitrogen), at 4 °C overnight. Thereafter, the membranes were washed with Tris‐buffered saline Tween buffer and incubated with HRP‐rabbit (ab191866, 1:5000, Abcam) or HRP‐mouse (bs-0296G-HRP, 1:1000, Bioss) conjugated secondary antibody for 1 h at room temperature (RT). An Odyssey Infrared Imaging System was used to scan the membranes for further analysis. β-actin (CL594-66009, 1:1000, Proteintech) was used as loading control. Band intensities were quantified using Image J software.
Bs 0296g hrp
Manufactured by Bioss Antibodies
Sourced in China
Bs-0296G-HRP is a horseradish peroxidase (HRP) conjugated secondary antibody. It is designed for use in various immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA) and western blotting, to detect and quantify target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bs 0295g hrp, by Bioss Antibodies (6 mentions)
Anti claudin 5, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
Ab51520, by Abcam (1 mentions)
Ab191866, by Abcam (1 mentions)
β actin, by Proteintech (1 mentions)
Anti p mtor 5536s, by Cell Signaling Technology (1 mentions)
Anti pi3k bs 10657r, by Bioss Antibodies (1 mentions)
Anti mtor 2983s, by Cell Signaling Technology (1 mentions)
Anti ampk 5832s, by Cell Signaling Technology (1 mentions)
Ab18258, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Ab93607, by Abcam (1 mentions)
Bs 2188r, by Bioss Antibodies (1 mentions)
Bs 0767r, by Bioss Antibodies (1 mentions)
Arg56625, by Arigo Biolaboratories (1 mentions)
Arg54150, by Arigo Biolaboratories (1 mentions)
Ac015, by ABclonal (1 mentions)
Anti occludin 13409 1 ap, by Proteintech (1 mentions)
10 protocols using bs 0296g hrp
1
Protein Expression Analysis by WB
2
Hippocampal Protein Expression Analysis
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Rat-isolated hippocampal regions were analyzed using Western blotting. Briefly, RIPA lysis buffer (Solarbio, Beijing, China) was used to homogenize the tissues. Total protein (50 μg) was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). Following incubation in blocking buffer (TBS with 5% nonfat milk and 0.1% Tween 20), the membranes were probed with primary antibodies directed against SHANK3 (1:1500, ab93607, Abcam, Cambridge, UK), GKAP (1:1000, ab93611, Abcam, Cambridge, UK), PSD95 (1:1000, ab18258, Abcam, Cambridge, UK), and GAPDH (1:2000, BS-2188 R, Bioss, Beijing, China). Goat anti-mouse IgG-HRP (1:3000, BS-0296G-HRP, Bioss) and goat anti-rabbit IgG-HRP (1:3000, BS-0295G-HRP, Bioss) were used as the secondary antibodies.
3
Multiplex Immunoblotting Assay for Cytokines
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Tissue proteins from each sample were denatured in 5× SDS-PAGE loading buffer and electrophoresis was performed on 12% tris-glysine agarose gels. The separated proteins were transferred to nitrocellulose membrane followed by blocking with 5% non-fat milk for 2 h at room temperature. The membrane was then incubated at 4°C over-night using primary antibodies for IFNg (ARG21502, Arigo), IL1a (ARG56667, Arigo), IL6 (ARG56625, Arigo), IL12a (bs-0767R, Bioss), IL12b (bs-10641R, Bioss), IL17a (ARG55256, Arigo), TNFa (ARG10158, Arigo), IL10 (ARG21475, Arigo), TGFb (ARG10002, Arigo), CXCR4 (ARG54674, Arigo), STAT3 (ARG54150, Arigo) and Tubulin (AC015, Abclonal). This was followed by incubation with horseradish peroxidase labeled secondary antibody (AS028, AS014, Abclonal; bs-0296G-HRP, Bioss) for 2 h at room temperature. Band intensity was measured by Tanon-5200 and Tanon MP.
4
Antibody Validation for Western Blot and Immunofluorescence
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For Western blot or immunofluorescence analysis, the following antibodies were used: anti-ADAM10 (A10438; ABclonal, Wuhan, China), anti-E-cadherin full length (3195; Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin NTF (A3044, ABclonal), anti-claudin-1 (ab129119; Abcam, Cambridge, MA, USA), anti-occludin (13,409-1-AP; Proteintech, Wuhan, China), anti-ZO-1 (21,773-1-AP; Proteintech), anti-HtrA1 (ab199529; Abcam), anti-MMP7 (10,374-2-AP; Proteintech), anti-FLAG (20,543-1-AP; Proteintech), and anti-GAPDH (10,494-1-AP; Proteintech). Horseradish peroxidase (HRP)-labeled goat anti-rabbit (bs-0295 G-HRP; Bioss, Beijing, China), HRP-labeled goat anti-mouse (bs-0296 G-HRP; Bioss), and Cy3-conjugated goat anti-rabbit (A0516; Beyotime) secondary antibodies were used.
5
Western Blot Protein Analysis Protocol
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Total protein was separated from cell lysates on ice using a radioimmunoprecipitation assay (RIPA) containing protease and phosphatase inhibitors (HEART, Xi’an, Shaanxi, China). We quantified protein levels using an Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were separated on 6 or 12% SDS-PAGE gels (Beyotime) and transferred to PVDF membranes (Millipore, Billerica, MA, USA), which were incubated with primary antibodies at 4 °C overnight. The primary antibodies used in this study were: γH2AX (1:1000, #80312, Cell Signaling Technology, Danvers, MA, USA), PTPRJ (1:5000, ab181244, Abcam, Cambridge, MA), and β-tubulin (1:10,000, AP0064, Bioworld, USA,). After washing four times with Tris-Buffered Saline and Tween 20 (TBST) buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:2500, bs-0295G-HRP, Bioss, Beijing, China) or goat anti-mouse immunoglobulin (Ig)G (1:2500, bs-0296G-HRP, Bioss, Beijing, China) for 1 h at room temperature was performed. The protein bands were visualized using chemiluminescence kits (Millipore, Billerica, MA, USA).
6
Protein Extraction and Western Blot Analysis
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To extract cellular protein, tissues were lysed by Pro-prep (catalog No. 17081; iNtRON Biotechnology, Korea), then homogenized by 20 strokes with a Dounce homogenizer, after which samples were centrifuged at 15,300 × g for 20 min at 4℃. Next, 50 µg of protein was loaded onto a 12.5% sodium dodecyl sulfate polyacrylamide gel, then transferred to nitrocellulose membrane (catalog No. IPVH00010; Millipore, USA). The blot was blocked with tris-buffered saline containing 0.5% tween-20 (TBS-T) including 5% skim milk for 1 h at room temperature (RT), then probed with the following primary antibodies at 4℃ overnight: anti-claudin-1 (1 : 1,000; 37-4900; Invitrogen, USA), anti-claudin-2 (1 : 1,000; 51-6100; Invitrogen), anti-claudin-4 (1 : 1,000; 36-4800; Invitrogen), anti-claudin-5 (1 : 1,000; 34-1600; Invitrogen), and anti-GAPDH (1 : 1,000; SC-137179; Santa Cruz Biotechnology, USA). After washing three times with TBS-T, the blots were treated with horseradish peroxidase-conjugated secondary antibody (bs-0296G-HRP; Bioss, USA; mouse IgG 1:3000 or sc-2004; Santa Cruz Biotech; rabbit IgG 1:3000) in TBS-T containing 5% skim milk for 2 h at RT. The blots were subsequently exposed to enhanced chemiluminescence reagent (SC-2048; Santa Cruz Biotech) and developed via GeneGnome5 (MOL5405; Syngene, USA). Signal specificity was confirmed through blotting without primary antibody.
7
Protein Quantification and Western Blot Analysis
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Concentrations of proteins extracted from radio immunoprecipitation assay (RIPA) Lysis Buffer (P0013B; Beyotime, Shanghai, China) for western blotting were measured using the bicinchoninic acid (BCA) protein assay kit (PC0021; Solarbio, Beijing, China). The membrane was incubated with the following secondary antibodies at room temperature for 1 h: goat anti-rabbit IgG heavy and light chains/horseradish peroxidase (H&L/HRP) (1:20 000, bs-0295G-HRP; Bioss, Beijing, China) and goat anti-mouse IgG H&L/HRP (1:20 000, bs-0296G-HRP; Bioss). An enhanced chemiluminescence (ECL) chemiluminescent substrate reagent kit (E412-01; Vazyme, Nanjing, China) was used to evaluate the blots. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to check the results.
8
Western Blot Analysis of Cellular Markers
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Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide gels. The resolved proteins were electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) for western blot analysis. Primary antibodies against AGEs (Abcam, ab23722, 1 : 2000), AMPK (ProteinTech, 66536-1-Ig, 1 : 1000), p-AMPK (Cell Signaling Technology, 2535 T, 1 : 1000), LC3 (ProteinTech, 14600-1-AP, 1 : 1000), p62 (ProteinTech, 18420-1-AP, 1 : 1000), Bax (ProteinTech, 23931-1-AP, 1 : 1000), Bcl-2 (ABclonal, A0208, 1 : 1000), and β-actin (Cell Signaling Technology, 4970S, 1 : 2000) were used according to the manufacturer's instructions. After the membranes were incubated overnight at 4°C, the membranes were washed three times with 1x TBST for 15 minutes. Subsequently, the membranes were incubated with goat anti-rabbit IgG (Bioss, bs-0295G-HRP, 1 : 2000) and goat anti-mouse IgG (Bioss, bs-0296G-HRP, 1 : 2000) for 1.5 h at room temperature and then washed 3 times with 1x TBST for 15 minutes. Finally, the membranes were exposed to X-ray film using an enhanced chemiluminescence system. The intensities of the bands were measured using the LabWorks 4.5 software.
9
Western Blot Analysis of Cellular Proteins
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Protein samples were quantified by Bradford assay using PRO-Measure solution (Intron, #21011) and subjected to protein electrophoresis (SDS-PAGE). Gels were blotted by wet transfer using a Bio-Rad Power Pac at 350 V for 1 or 2 h. Membranes were blocked and incubated with primary antibodies for overnight at 4 ℃. After washing, the membranes were incubated with secondary antibodies overnight at 4 °C. Next, after a washing step, the protein bands were detected using an ECL kit (XLS025-0000, Cyanagen) on the ChemiDoc system (Fusion Solo, Vilber Lourmat).
The primary antibodies used were: β-actin [Rabbit polyclonal antibody (Rab Poly Ab), Santa Cruz, sc-130656], HK1, HK2, PKM2, PDH [Rab Poly Ab, Cell Signaling Technology (CST), #8337], eIF2α (Rab Poly Ab, CST, #9722), peIF2α [Rabbit monoclonal antibody (Rab mono Ab), CST, #3597], CHOP (Mouse Monoclonal antibody, Invitrogen, #MA1-250), ANP (Rab poly Ab, Abcam, ab14348), SPT1 (Rab poly Ab, ABclonal, A6750), and PGRMC1 (Rab mono Ab, CST, #13856). The secondary antibodies used were: Mouse anti-rabbit IgG (211-032-171, Jackson ImmunoResearch, 1:5000) and Goat anti-mouse IgG antibody (BS-0296G-HRP, Bioss, 1:5000).
The primary antibodies used were: β-actin [Rabbit polyclonal antibody (Rab Poly Ab), Santa Cruz, sc-130656], HK1, HK2, PKM2, PDH [Rab Poly Ab, Cell Signaling Technology (CST), #8337], eIF2α (Rab Poly Ab, CST, #9722), peIF2α [Rabbit monoclonal antibody (Rab mono Ab), CST, #3597], CHOP (Mouse Monoclonal antibody, Invitrogen, #MA1-250), ANP (Rab poly Ab, Abcam, ab14348), SPT1 (Rab poly Ab, ABclonal, A6750), and PGRMC1 (Rab mono Ab, CST, #13856). The secondary antibodies used were: Mouse anti-rabbit IgG (211-032-171, Jackson ImmunoResearch, 1:5000) and Goat anti-mouse IgG antibody (BS-0296G-HRP, Bioss, 1:5000).
10
Western Blot Analysis of Cell Adhesion and Exosomal Markers
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An equal amount of protein extract was separated on 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were cultured with the primary antibodies at 4°C overnight, and then with the secondary antibody for 2 h. Next, the membranes were developed and visualized using the enhanced chemiluminescence reagent (Millipore). The protein bands were analyzed using Quantity One (Bio-Rad Laboratories, Hercules, CA, United States). The primary antibodies used in this study were as follows: mouse monoclonal antibody N-cadherin (1:2,000, 66219-1-Ig, Proteintech), mouse monoclonal antibody E-cadherin (1:2,000, 60335-1-Ig, Proteintech), mouse monoclonal antibody vimentin (1:10,000, 60330-1-Ig, Proteintech), mouse monoclonal anti-CD9 (1:2,000, 60232-1-Ig, Proteintech), mouse monoclonal anti-CD81 (1:3,000, 66866-1-Ig, Proteintech), mouse monoclonal antibody GRP94 (1:1,000, 60012-1-Ig, Proteintech), rabbit polyclonal antibody PPAPDC1A (1 μg/mL, PA5-20864, Invitrogen), and mouse monoclonal antibody GAPDH (1:20,000, 60004-1-Ig, Proteintech). The secondary antibodies used in this study were horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:2,000, bs-0296G-HRP, Bioss, Beijing, China) and HRP-labeled goat anti-rabbit IgG (1:2,000, bs-0295G-HRP, Bioss).
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