Proteins were extracted from the jejunum and ileum samples and IPEC-J2 cells, as previously described (Zhang et al., 2015 (link); Yang et al., 2017 (link)). The primary antibodies were rabbit anti-phospho-Ser473 (p)-Akt (1:1000, ab138726), rabbit anti-phospho-Tyr1068 (p)-EGFR (1:250, ab32430; Epitomics, Burlingame, CA), rabbit anti-LC3A/B (1:1000, Cell Signaling Technology, Danvers, MA), rabbit anti-total-EGFR (1:500, 18986-1-AP), rabbit anti-total-Akt (1:1000, 10176-2-AP), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:500, 60004-1-Ig, Proteintech Group, Chicago, IL). Horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG (1:5000, SA00001-1; Proteintech Group) or goat anti-rabbit IgG (1:5000, SA00001-2; Proteintech Group) were used as secondary antibodies.
Affinipure goat anti mouse igg
Manufactured by Proteintech
Sourced in United States, China
AffiniPure goat anti-mouse IgG is a laboratory reagent used for the detection and quantification of mouse immunoglobulin G (IgG) in various immunoassay and research applications. It is an affinity-purified secondary antibody produced in goats and specifically designed to recognize and bind to mouse IgG molecules.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Proteintech (6 mentions)
Sa00001 2, by Proteintech (5 mentions)
Sa00001 1, by Proteintech (5 mentions)
Anti β actin, by Proteintech (3 mentions)
Bca method, by Beyotime (2 mentions)
Ripa lysis buffer, by CoWin Biotech (2 mentions)
Anti phospho iκbα, by Cell Signaling Technology (2 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (2 mentions)
Anti nf κb, by Cell Signaling Technology (2 mentions)
Anti phospho p38, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Affinipure goat anti rabbit igg, by Proteintech (2 mentions)
Rabbit anti lc3a b, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Thermo Fisher Scientific (1 mentions)
B 1235, by Vector Laboratories (1 mentions)
Ab178876, by Abcam (1 mentions)
Anti n cadherin, by Santa Cruz Biotechnology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
16 protocols using affinipure goat anti mouse igg
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis of MGC803 Cells
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The total proteins from MGC803 cells and MGC803 sphere cells were extracted with a lysis buffer. The concentration of proteins in the supernatant was analyzed using the BCA method (Beyotime, China). The protein samples (60 μg per lane) were loaded onto 10% SDS-polyacrylamide gels, electrophoresed, and then transferred to nitrocellulose membranes that were first blocked with 10% (wv-1) non-fat milk in TBST [50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween-20] at room temperature for 1 h and then incubated with a primary antibody diluted in TBST (anti-LGR5 mouse mAb, 1:2000, OriGene; anti-beta-actin mAb, 1:5000, Proteintech) overnight at 4°C. After washing three times with TBST, the membranes were incubated with a peroxidase-conjugated Affinipure Goat Anti-Mouse IgG, 1:5000, Proteintech) for 1.5 h at room temperature. The bands were visualized and quantified using the ECL chemiluminescence detection system (FluorChem E, Alpha, USA).
3
Immunoblotting Analysis of EMT Markers
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Cells in experimental and control groups were lyzed and centrifuged. After determined the protein concentrations, the cell lysate samples (30 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto NC membranes. The membranes were blocked with 5% BSA in TBST and immunoblotted by the primary antibodies. The primary antibodies used in this study were:anti-GALNT6 (1:1000, sc-100755, Santa Cruz Biotechnology), anti-E-cadherin (1:500, 13-1700, Invitrogen), anti-N-cadherin (1:200, sc-393933, Santa Cruz Biotechnology), anti-Slug (1:500, ab27568, Abcam), anti-GRP78 (1:1000, ab21685, Abcam), anti-phospho-MEK1/2 (1:1000, #9154, CST), anti-MEK1/2 (1:1000, ab178876, Abcam), anti-phospho-ERK1/2 (1:1000, #4370, CST), anti-ERK1/2 (1:1000, #4695, CST), biotinylated anti-Viciavillosa agglutinin (specific to GalNAc-Ser/Thr) (VVA, 1:1000, B-1235, Vector) and anti-GAPDH (1:2000, 10494-1-AP, Proteintech). The secondary antibodies were HRP-conjugated Affinipure Goat Anti-Mouse IgG (1:5000, SA00001-1, Proteintech), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (1:5000, SA00001-2, Proteintech) and HRP-streptavidin (1:10000, SA-5014, Vector). The protein bands were visualized by enhanced chemiluminescent reagents (Advansta) and the intensity of protein band was quantified by ImageJ software.
4
Western Blot Analysis of Protein Signaling
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The cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (CoWin Biosciences, China) with 100 U of proteinase inhibitors (CoWin Biosciences, China) and 20 μM NaF on ice for Western blotting assay. Primary antibodies used in this study were anti-PDCoV N (1:5, provided by Professor Pinghuang Liu, China Agricultural University, Beijing, China), anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38, anti-phospho-NF-κB p65 (1:500, Cell Signaling Technology, USA), anti-phospho-IκBα (1:300, Cell Signaling Technology, USA), anti-ERK, anti-JNK, anti-p38, anti-IκBα, anti-NF-κB (1:1000, Cell Signaling Technology, USA) and anti-β-actin (1:1000, ProteinTech Group, USA). Horseradish peroxidase conjugated to AffiniPure goat anti-mouse IgG (1:5000, ProteinTech Group, USA) or goat anti-rabbit IgG (1:5000, ProteinTech Group, USA) was used as secondary antibodies.
5
Protein Extraction and Western Blotting for Subcellular Analysis
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We used the same method as before to perform Western blotting.37 Before Western blotting, we used a total protein extraction kit (BC3710, Solarbio), cellular mitochondrial isolation kit (C3601, Beyotime) or tissue mitochondrial isolation kit (C3606, Beyotime), and nuclear protein extraction kit (R0050, Solarbio) to extract total protein, mitochondrial protein or de‐mitochondrial cytoplasmic protein, and nuclear protein, respectively. The primary antibodies used were anti‐PARP‐1 (1:1000, DF7198, Affinity); anti‐AIF (1:1000, BF0591, Affinity); anti‐COX IV (1:1000, AF5468, Affinity); anti‐Histone H3 (1:1000, AF0863, Affinity); anti‐β‐actin (1:10,000, 66009‐1‐Ig, Protentech); anti‐LC3BI/II (1:1000, A5402, Affinity); anti‐PINK1 (1:200, PA5‐85930, Invitrogen); anti‐Parkin (1:200, 702785, Invitrogen); anti‐SIRT3 (1:1000, Affinity, AF5135); anti‐SOD2/MnSOD (acetyl K68) (1:5000, ab137037, Abcam); and anti‐SOD2/MnSOD (1:5000, ab13533, Abcam). The second antibodies used were HRP‐conjugated AffiniPure Goat Anti‐Mouse IgG (1:10,000, Proteintech) and HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG (1:10,000, Proteintech). Finally, PVDF molds were visualized on a Tanon 2500R gel imaging system (Tanon) using a developer (Tanon), and the band intensity was quantified using ImageJ 1.39V software.
6
Validating TMT Proteomics with Western Blot
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Western blot was carried out to validate the TMT-based proteomics analysis results. Skeletal muscle total protein was extracted with RIPA lysis buffer containing protease and phosphatase inhibitors, and the concentrations were determined using a BCA protein assay kit (P0010S, Beyotime, China). The total protein (10 μg) from each sample was separated by SDS-PAGE gel electrophoresis and then electrotransferred onto PVDF membranes. After 1.5 h of blocking in the blocking solution, the PVDF membrane containing the proteins was incubated in the desired primary antibody, including Anti-ApoA1 antibody (1:1000, ab227455, Abcam, UK), Anti-ApoA4 antibody (1:10,000, 5700S, Cell Signaling Technology, USA) and Anti-alpha Tubulin antibody (1:5000, ab7291, Abcam, UK) overnight at 4 ℃. After incubating with HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (1:5000, SA00001-1, Proteintech, USA) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:5000, SA00001-2, Proteintech, USA) for 1 h, PVDF membrane was washed with TBST, incubated with an electro-chemiluminescence (ECL) developing solution, and exposed and photographed. Using ImageJ, target protein bands are measured and their relative levels of expression are quantified by the ratio of the corresponding protein to α-tubulin.
7
Antibody Characterization for Neuronal Studies
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The mouse antibodies used were as follows: anti-NeuN (Ab104225, Abcam, dilution IF: 1:500). Rabbit antibodies used in this study were as follows: anti-NRXN1 antibody was used (GTX54845, GeneTex, dilution, IF:1:30, IHC:1:30; Santa Cruz, sc136001, WB:1:100), anti-MAP 2 (Ab32454, Abcam, dilution IF: 1:500), anti-GFP (AF5066, Beyotime, dilution IF: 1:100), and anti-GAPDH (10494, Proteintech, dilution WB 1:1000). Other antibodies were as follows: Donkey polyclonal Secondary Antibody to Mouse IgG (Alexa Fluor594, ab150108, Abcam, dilution IF 1:1000), Donkey polyclonal Secondary Antibody to Rabbit IgG (Alexa Fluor488, ab150061, Abcam), HRP-conjugated AffiniPure Goat Anti-Mouse IgG(H+L) (SA00001-1, Proteintech, dilution WB:1:5000), and HRP-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) (SA00001-2, Proteintech, dilution WB:1:5000).
8
Comprehensive Western Blot Analysis
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Western blot protocol was performed as previously described.22 (link) Primary antibodies included anti-ACOT7 (1:7000, 15972-1-AP, Proteintech, China), anti-KLF13 (1:1000, 18352-1-AP, Proteintech, China), anti-GAPDH (1:7000, 10494-1-AP, Proteintech, China), anti-β-tubulin (1:5000, #M20005, Abmart), anti-cyclin D1 (1:1000, 26939-1-AP, Proteintech, China), anti-CDK2 (1:1000, 10122-1-AP, Proteintech, China), anti-CDK4 (1:1000, 11026-1-AP, Proteintech, China), anti-E-cadherin (1:7000, 20874-1-AP, Proteintech, China), anti-N-cadherin (1:7000, 22018-1-AP, Proteintech, China) and anti-vimentin (1:5000, ab92547, Abcam). The secondary antibodies included HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) (1:7000, SA00001-2, Proteintech, China) and HRP-conjugated Affinipure goat anti-mouse IgG (H+L) (1:7000, SA00001-1, Proteintech, China).
9
Western Blot Analysis of Cell Signaling Proteins
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Tissues and cells were lysed in RIPA lysis buffer (Catalog No. P0013B, Beyotime). The lysis was centrifuged, and the supernatant was collected for Western blot analysis. Samples of protein were resolved on SDS‐PAGE and transferred onto PVDF membranes (Catalog No. ISEQ00010, Millipore). The membranes were incubated with the following primary antibodies overnight at 4°C: anti‐Hic‐5 (Catalog No. ab42476, 1:1000, abcam), anti‐α‐SMA (Catalog No. ab5694, 1:500, abcam), anti‐Col1a1 (Catalog No. GB11022, 1:1000, Servicebio), anti‐IL‐6 (Catalog No. GB11117, 1:1000, Servicebio), anti‐NF‐κB/p65 (Catalog No. 10745‐1‐AP, 1:1000, Proteintech), anti‐Vimentin (Catalog No. GB11192, 1:1000, Servicebio) and anti‐GAPDH (Catalog No. 60004‐1‐Ig, 1:5000, Proteintech). Then, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. The following secondary antibodies conjugated to horseradish peroxidase: Affinipure goat antimouse IgG (Catalog No. SA00001‐1, 1:5000, Proteintech) and Affinipure goat anti‐rabbit IgG (Catalog No. SA00001‐2, 1:5000, Proteintech).
10
Serum-based IgG Determination
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The sera were collected from each immunized mouse 10 day after the last immunization to determine specific IgG using cell-based ELISA. The serum was diluted 50-fold, followed by separation into eight gradient dilutions at 1:4 ratios. HeLa cells transfected with the individual glycan deletion GPC or WT GPC were served as antigens, which were blocked, washed, and incubated with serums, followed by detection with HRP-conjugated AffiniPure Goat Anti-Mouse IgG (Proteintech, Wuhan, China).
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