The concentration of protein was determined by BCA method to prepare protein loading samples. The brain protein extract (30 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to the PVDF membrane. The membrane was blocked with 5% skim milk on a shaker for 1 h. After washing with TBST, the membranes were incubated with the primary antibodies rabbit anti-MBP (1:500, Abcam), goat anti-Iba-1 (1:500, Abcam), rabbit anti-Arg-1 (1:800, Cell Signaling Technology), rabbit anti-iNOS(1:500, Genetex), rabbit anti-BDNF (1:500, Abclonal), rabbit anti-GDNF(1:500, Abcam), rabbit anti-CNTF (1:1000, Abcam), rabbit anti-NG2 (1:1000, Abcam), rabbit anti-β-actin (1:1000, Abclonal) and rabbit anti-Tubulin (1:1000, Abclonal) overnight at 4 °C. After washing with TBST, the membranes were added with the HRP–conjugated goat anti-rabbit (1:3000, Abclonal) and rabbit anti-goat (1:1000, Boster) second antibodies for 2 h at RT. After washing with TBST, immunoblots were developed with an enhanced chemiluminescence systemand measured using Quantity Software (Bio-Rad, Hercules, CA, USA).
Goat anti rabbit
Manufactured by Boster Bio
Sourced in China, United States
Goat anti-rabbit is a secondary antibody used in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry. It is produced by immunizing goats with rabbit immunoglobulins, resulting in the generation of antibodies that specifically recognize and bind to rabbit antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse, by Boster Bio (10 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Solabio (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Rabbit anti ng2, by Abcam (1 mentions)
Rabbit anti gdnf, by Abcam (1 mentions)
Rabbit anti mbp, by Abcam (1 mentions)
Rabbit anti inos, by GeneTex (1 mentions)
Rabbit anti bdnf, by ABclonal (1 mentions)
Rabbit anti tubulin, by ABclonal (1 mentions)
Rabbit anti arg1, by Cell Signaling Technology (1 mentions)
Goat anti iba1, by Abcam (1 mentions)
Rabbit anti β actin, by ABclonal (1 mentions)
Quantity software, by Bio-Rad (1 mentions)
Ecl detection system, by Bio-Rad (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Boster Bio (1 mentions)
Non fat milk, by Sangon (1 mentions)
Gel analysis v 2, by Clinx (1 mentions)
13 protocols using goat anti rabbit
1
Western Blot Analysis of Neural Markers
2
Immunohistochemical Analysis of Bone Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry (IHC) was performed with reference to a previous procedure [27 (link)]. The primary antibodies used were goat anti-rabbit osteocalcin (OCN; 1:400), runt-related transcription factor 2 (RUNX2; 1:200), β-catenin (1:300), and PTH1R (1:300), and anti-mouse matrix metalloproteinase 9 (MMP9; 1:200), vascular endothelial growth factor (VEGF; 1:200), and goat anti-rat BrdU (1:200). All antibodies were purchased from Santa Cruz Biotechnology Corporation (Santa Cruz, CA, USA). The secondary biotinylated goat anti-mouse, rabbit anti-goat, and goat anti-rabbit IgGs were purchased from Boster (Wuhan, China). Positive expression cells were observed the region of interest with an Olympus microscope, and five random regions within the observation range were selected and counted. Results were identified by positive cell values and standard deviations and used for statistical analysis.
3
Quantifying SLC26A6 Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were dissociated using radio immunoprecipitation assay and phenylmethanesulfonyl fluoride. The protein (40 µg/lane) was electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Immobilon-P Transfer Membrane; Millipore Corporation, Burlington, MA, USA). The protein samples were equally mixed, and the expression of SLC26A6 in the two groups was analyzed. The primary antibody was goat anti-SLC26A6 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse β-actin (1:500; Boster, Wuhan, China). The secondary antibody was rabbit anti-goat (1:5000; Boster, Wuhan, China) or goat anti-mouse (1:5000; Boster, Wuhan, China). After incubation with the secondary antibody at room temperature for 2 h, proteins were detected with the Bio-Rad Clarity Western Enhanced Chemiluminescence (ECL) Substrate (Bio-Rad Laboratories, Hercules, CA, USA) and ECL detection system (1705061; Bio-Rad Laboratories, Hercules, CA, USA). The immunohistochemical (IHC) assays were performed according to the standard protocol, and the dilution rate of anti-SLC26A6 primary antibody was 1:100. Microscopy (BX53; Olympus, Tokyo, Japan) was used to observe the expression of SLC26A6. Fluorescence intensities were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
4
Quantitative Western Blot Analysis of Inflammatory Markers
5
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues or cells were treated with lysis buffer containing 1% PMSF. Protein quantitation was determined using the bicinchoninic acid assay. Thirty micrograms of protein was run on 10% SDS‐PAGE gel. Subsequently, the gel‐separated proteins were transferred onto PVDF membranes by wet transfer. The membranes were blocked with 5% non‐fat milk for 2 h at RT and then incubated overnight with the primary antibody at 4°C, followed by incubation with goat anti‐mouse (Boster Biological Technology Co. Ltd, Cat no: BA1075, 1:2000) or goat anti‐rabbit (Boster Biological Technology Co. Ltd, Cat no: BA1054, 1:2000) IgG‐HRP secondary antibody, for 1 h at RT. Immunoblots were visualised using a chemiluminescence kit (Millipore). β‐actin or GAPDH was selected as the internal control for gene expression normalisation based on the molecular weight of protein. The primary antibodies and corresponding secondary antibodies used in present study and their appropriate working dilutions are listed in Table 2 .
6
Protein Isolation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular protein was isolated by lysis in RIPA buffer (Boster, Wuhan). Proteins in all samples were quantified with Bicinchoninic acid protein assay. Proteins of equal amounts from all samples were separated with SDS/PAGE gel (Bio-Rad, United States) and transferred onto PVDF membrane (Bio-Rad, United States). Bands were sealed with 5% skim milk, incubated in primary antibodies at 4°C overnight, then in secondary antibodies at room temperature for 1 h, and then examined and analyzed by using ChemiDoc™ XRS+ with Image Lab™ Software (Bio-Rad, United States). Following antibodies were purchased: anti-caspase-3 (#9662), anti-PARP (#9532), anti-Phospho-SAPK/JNK (Thr183/Tyr185) (#4688), anti-Phospho-p38 MAPK (Thr180/Tyr182) (#4511), anti-HDAC1 (#34589), anti- HDAC2 (#57156), and anti-Histone H3 (#4499) were purchased from Cell Signaling Technology (Beverly, MA, United States); anti-GAPDH (60004-1-Ig) was purchased from Proteintech Group (Chicago, IL, United States); anti-Phospho-ERK (AP0472), anti-Phospho-MEK (AP1021) and anti-CDK6 (A0705) were purchased from ABclonal Technology (Wuhan, China); anti-cyclin D1 (YT1172) was purchased from Immunoway Biotechnology Company (TX, United States); goat anti-rabbit and goat anti-mouse were obtained from Boster Biological Technology (Wuhan, China).
7
Western Blot Analysis for Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from the cells and tissues using radio immunoprecipitation assay buffer (BIOSS) mixed with phenylmethylsulfonyl fluoride (BIOSS) and quantified using bicinchoninic acid assay (Beyotime Institute of Biotechnology). Protein extractions (30ug per well) were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (MilliporeSigma). The membranes were blocked with 5% fat-free dried milk for 1 h at room temperature. After incubation with high-affinity anti-FTO (1:1000, Abcam, USA), anti-STAT3 (1:1000, Cell Signaling Technology, USA), anti- phosphorylation-STAT3 (1:1000, Cell Signaling Technology, USA), anti-Bcl-2 (1:1000, Cell Signaling Technology, USA), anti-c-Myc (1:1000, Abways Technology, China), anti-CyclinD-1 (1:1000, Abways Technology, China), anti-β-actin (1:5 000, Bioss, China), or anti-GAPDH (1:5000, Bioss, China) antibodies at 4°C overnight, the membranes were incubated with the HRP-conjugated secondary antibodies goat anti-rabbit (1:10,000; Boster, China) or goat anti-mouse (1:5000; Boster, China) for 1 h at room temperature. Proteins were detected using BeyoECL chemiluminescence kit (Biyuntian, China) and detected using the Amersham ImageQuant 800 system (Cytiva). The density of bands were measured using ImageJ (v1.51, National Institutes of Health).
8
BDNF Protein Quantification in Hippocampus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hippocampus tissues and hippocampal neurons were treated with ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) to extract protein. After determining the protein concentrations using BCA Assay Kit (Beyotime), protein samples were separated in SDS-PAGE gels and then transferred onto PVDF membranes (Invitrogen). Subsequently, the membrane was incubated with skim milk, primary antibody against BDNF (1:2000, Boster, Beijing, China) or GAPDH (1:2000, Boster), and secondary antibody (Goat anti-rabbit, 1:10,000, Boster) in turn. The signal intensity was determined by Super ECL Detection Reagent (Yeasen, Shanghai, China).
9
Western Blot Analysis of p-AKT and AKT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell proteins were extracted at 4 °C using RIPA lysis buffer (Solabio, Beijing, China) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Proteins were resolved using 8%-12% SDS polyacrylamide electrophoresis and electro-transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). Then we blocked the blots with 5% non-fat milk for 1 h at room temperature. Western blots were probed with antibodies against p-AKT (Ser473, 1:1000, CST, USA) and total AKT (1:1000, CST, USA), both from Cell Signaling Technology), GAPDH (1:1000, CST, USA) at 4°C overnight. After washing, the blots were then incubated with the secondary antibody, goat anti-mouse (1:2000) and goat anti-rabbit (1:2000) (Boster Biological Technology co., ltd, USA) for 2 h at room temperature. Western blotting was performed at least three biological replicates.
10
Profiling of Inflammasome Pathway in HUVEC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from HUVECs using Cell lysis buffer for Western and IP [20mM Tris(pH7.5), 150mM NaCl, 1% Triton X-100, Beyotime, China]. The concentrations of protein were determined by a BCA protein assay kit (Vazime, China). 20 μg proteins were separated by BeyoGel™ Plus Precast PAGE Gel (Beyotime, China) and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% dry milk, the membrane was incubated with primary antibodies against NLRP3 (1:1000, AdipoGen, USA), ASC (1:1000, AdipoGen, USA), Caspase-1 (1:1000, Abcam, UK), GSDMD (1:500, Santa, USA), and IL-1β (1:500, Santa Cruz, USA), AMPKα (1:1000, Cell Signaling Technology, USA), and Phospho-AMPKα (Thr172) (1:1000, Cell Signaling Technology, USA), β-actin (1:2000, Proteintech, China) overnight at 4°C. Following incubation, the membrane was washed thrice with TBST (0.05% Tween 20). After incubation with the corresponding secondary antibody [goat anti-rabbit (1:5000, BOSTER Biological Technology co. ltd, China) and goat anti-mouse (1:5000, BOSTER Biological Technology co. ltd, China)], the membrane was exposed to an enhanced chemiluminescence kit (Vazime, China), and observed using a Clinx ChemiScope 3500 (Clinx Science instrument Co. Ltd, China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!