Protein samples were harvested from cells, and were tested and treated with polyacrylamide gels electrophoresis, and then transferred to PVDF membrane. The membrane was hybridised first with Rabbit polyclonal HDGF antibody (ab176697, Abcam, USA) or Rabbit polyclonal GAPDH (ab9485, Abcam, USA) and then hybridised with secondary Goat Anti-Rabbit antibody (A0208, Beyotime, China) alternately. After that, the membrane was incubated with BeyoECL Plus reagent (Beyotime, Nanjing, China), and the images were obtained and analysed to quantify the expression of HDGF protein.
Goat anti rabbit antibody
Manufactured by Beyotime
Sourced in China, United States
The Goat anti-rabbit antibody is a laboratory reagent used in various immunological techniques. It is a secondary antibody that specifically binds to rabbit primary antibodies, allowing for the detection and analysis of target proteins or molecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Beyoecl plus reagent, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
A0208, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Ecl plus reagent, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Protein lysis buffer, by Beyotime (1 mentions)
Chemidoc touch system, by Bio-Rad (1 mentions)
Goat anti mouse antibody, by Beyotime (1 mentions)
Pierce ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti lc3, by Merck Group (1 mentions)
Rabbit anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Beyotime (1 mentions)
Horseradish peroxidase conjugated goat anti mouse antibody, by Santa Cruz Biotechnology (1 mentions)
Gapdh antibody, by Thermo Fisher Scientific (1 mentions)
Ecl substrate, by Beyotime (1 mentions)
12 protocols using goat anti rabbit antibody
1
Western Blot Analysis of HDGF Protein
2
Western Blot Analysis of DHCR7 in Cervical Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cervical cancer cells were cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Inc., MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Inc., MA, USA). Cells were incubated at 37°C in a 5% CO2 humidified incubator. Protein lysis buffer (Beyotime, Shanghai, China) was used to extract the cell lysates and bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China) was used to determine the protein concentration. The protein samples were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (EMD Millipore, MA, USA). 5% bovine serum albumin (Beyotime, Shanghai, China) was used to block the membranes. Rabbit anti-DHCR7 antibody (1 : 2000, Invitrogen) and rabbit anti-β-actin were used to react with the protein samples at 4°C overnight. Then, goat anti-rabbit antibody (1 : 1000, Beyotime, Shanghai, China) was used to incubate the membranes at room temperature for 1 h. ECL plus reagent (Beyotime, Shanghai, China) was used to detect the brands.
3
Western Blot Analysis of Tight Junction Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were harvested and lysed on ice by RIPA Lysis Buffer (Beyotime, P0013B) containing protease inhibitor (Beyotime, ST506). And the lysates were centrifuged at 10,000 × g at 4 °C for 15 min to remove the cell debris. The concentration of the whole cellular lysis was quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher, 23225) following the manufacturer’s instructions. Cell lysates were heat-denatured and separated by SDS-PAGE, blotted onto PVDF membranes, which were blocked with 5% skim milk rotating for 2 h at room temperature. The membrane was probed against the target proteins by incubation with primary antibody of Rabbit anti-CLDN8 (1.0 μg/ml, Novus Biologicals, NBP1-59157), Rabbit anti-CLDN4 (1:1,000, Abcam, ab53156), Mouse anti-Flag (0.1 μg/ml, Medical & Biological Laboratories, M185), or Mouse anti-β-actin (1:5,000, CUSABIO BIOTECH, CSB-MA000187), rotating overnight at 4 °C, subsequently secondary Goat anti-Rabbit antibody (1:10,000, Beyotime, A0208) or Goat anti-Mouse antibody (1:10,000, Beyotime, A0216) coupled to horseradish peroxidase rotating for 1 h at room temperature. Then the bands were visualized using the Pierce ECL Western blot substrate (Thermo Fisher, 32209) and the images were taken by using the Bio-Rad ChemiDoc Touch system (Bio-Rad).
4
Western Blot Analysis of Immune Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was utilized to analyze the expression of PI3KR1, Atg9A, and LC3 proteins. A549 cells were seeded in 1% DMED/F‐12 at a concentration of 5 × 105 cells per well in 6‐well plates. Cells were then infected with the OMVs (200 µg mL−1) and/or SLAP‐S25 (20 µg mL−1) at 37 ˚C for 6 h. The primary antibodies included rabbit anti‐ PI3KR1 (ABclonal Technology Co., Wuhan, China), rabbit anti‐Atg9A (Cell Signaling Technology, US), rabbit anti‐LC3 (Sigma, US), and rabbit anti‐β‐actin antibodies (MBL International, US). The secondary antibody was a goat anti‐rabbit antibody (Beyotime Biotechnology Co., Shanghai, China). The gray values of protein bands were quantified by ImageJ software.
5
Western Blot Analysis of IFNγR1 in THP-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts from the THP-1 cells were prepared using a modified radioimmunoprecipitation buffer with 0.5% sodium dodecyl sulfate in the presence of proteinase inhibitor cocktail. Protein weighing 50 µg was electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis minigels and transferred onto polyacrylamide fluoride membranes. After blocking with 5% bovine serum albumin, the membranes were incubated with mouse anti-IFNγR1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Beyotime, Shanghai, China) at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse antibody (Santa Cruz, CA, USA) or goat anti-rabbit antibody (Beyotime, Nantong, China) for 1 h at room temperature. Signals were developed using a chemoluminescent substrate (Amersham Biosciences Corp., Piscataway, NJ, USA) and visualized through X-ray films. The gray value of each specific band on the images was digitized using Image J analysis software (NIH, Bethesda, MD, USA). Protein gray values were divided by the internal reference GAPDH to correct errors. Results of a sample represent the relative protein content.
6
Western Blot Quantification of GPAM Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 48 h after transfection, RIPA lysis buffer (Beyotime, China) was utilized to lyse the cells. The total protein was then quantified with a BCA kit (Sangon, China). The 20 μg of protein was separated by 4–20% precast gel (Willget, China), and then transferred to a nitrocellulose membrane (PALL, USA). The membrane was then blocked by 3% skim milk (Beyotime, China) at 37°C for 45 min, followed by incubation with GPAM antibody (1:100) (Invitrogen, USA) or GAPDH antibody (1:5,000) (Invitrogen, USA) at 4°C overnight. After being incubated with goat anti-rabbit antibody (1:2,000) (Beyotime, China) at 37°C for 90 min, the membrane was incubated with ECL substrate (Beyotime, China), and exposed with X-ray film (Kodak, USA). Quantification of protein bands was performed by ImageJ 1.8.0 (NIH, USA).
7
Quantifying Gene and Protein Expression in Placental Vessels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from human placental vessels (HPV) using Trizol reagent (Invitrogen) according to manufacturer's instructions, and was then reversed transcribed using the first-strand cDNA Synthesis Kit (Toyobo Corp., Shanghai, China). qRT-PCR was performed using SYBR Green Supermix Taq Kit (Takara Biotechnology Co., Ltd., Dalian, China) and analyzed on an iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primer sequences were listed in Supplementary Table S2. ∆∆Ct method was used to comparatively quantify the amount of mRNA levels. The protein abundance of AVPR1A, AVPR2, and PKC (α, and β) in HPV was measured with Western blot normalized to β-actin. The primary antibodies were the rabbit polyclonal antibody (Santa Cruz Biotechnology) against AVPR1A, AVPR2, PKCα, PKCβ and β-actin (all 1:1000). The secondary antibody was the goat anti-rabbit antibody (1:1000; Beyotime Biotechnology, Jiangsu, China). Immuno-signals were visualized using UVP imaging system (Tianneng, Shanghai, China). Imaging signals were calculated and analyzed, and then the ratio of band brightness to β-actin was acquired to measure the relative protein expression level. Analyses was performed as previously described [25 (link),26 (link)].
8
Hepatocyte Lipid Visualization and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hepatocytes were fixed by using 4% paraformaldehyde for 20 min and treated with the immunol staining blocking buffer (Beyotime, Shanghai, China) at 4°C for 1 h. LC3 antibody (16/14 kDa; Rabbit Anti-LC3B antibody-N-terminal; 1:1,000; Abcam, Cambridge, United Kingdom) was added to the samples, then the sections were incubated at room temperature for 2 h. Afterward, the sections were stained with a fluorescent secondary antibody (1:500 dilution, goat anti-rabbit antibody; Beyotime, Shanghai, China) and treated with 3.8 μmol/L BODIPY 493/503 (D3922, ThermoFisher Scientific, Waltham, MA), a staining tracer for neutral lipids, for 10 min in the dark (Lee et al., 2014 (link)). The nucleus was stained with DAPI (C0065, Solarbio, Beijing, China) through binding to adenine-thymine-rich regions of DNA (Wang et al., 2019 (link)). Hepatocytes were visualized via a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) in order to determine the intensity of fluorescence of stained hepatocytes.
9
Protein Expression Analysis of NAMPT and NMRK1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was utilized to analyze the expression of NAMPT and NMRK1. A549 cells were seeded in antibiotic-free medium at a concentration of 2 × 106 cells per well in 6-well plates. Cells were then infected with the S. chromogenes isolate SC10 and/or Av. paragallinarum isolate X1-1S-1 at an MOI of 1 at 37°C for 6 h. The primary antibodies included rabbit anti-NAMPT (Invitrogen, Thermo Fisher Scientific, US), rabbit anti-NRK1 (Abcam Co., UK) and rabbit anti-β-tubulin antibodies (Cell Signaling Technology, US). The secondary antibody was a goat anti-rabbit antibody (Beyotime Biotechnology Co., Shanghai, China). Gray values of protein bands were quantified by ImageJ software.
10
Molecular Markers of Podocyte Injury
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nephrin primary antibody, podocin primary antibody, CD2-associated protein (CD2AP) primary antibody, and desmin primary antibody were purchased from Abcam, UK. Goat anti-rabbit antibody was purchased from Beyotime Biotechnology Corporation, China, and β-actin antibody was purchased from Santa Cruz Biotechnology, Inc., USA. Trizol reagent was purchased from Ambion Corporation, China. Nephrin, podocin, CD2AP, and desmin Taqman were purchased from ABI, USA. An EasyScript First-Strand cDNA Synthesis SuperMix kit was purchased from TransGen Biotech, China.
Electrophoresis apparatus (DYCP-40) was purchased from the Beijing Liuyi Instrument factory. A Hitachi H-600 transmission electron microscope was obtained from Hitachi. A Mindary BS480 analyzer and a COBAS701 biochemical analyzer were used. An ABI 7900HT high-throughput real-time fluorescence quantitative real-time PCR (qPCR) system (Applied Biosystems, USA) was used in this study.
Electrophoresis apparatus (DYCP-40) was purchased from the Beijing Liuyi Instrument factory. A Hitachi H-600 transmission electron microscope was obtained from Hitachi. A Mindary BS480 analyzer and a COBAS701 biochemical analyzer were used. An ABI 7900HT high-throughput real-time fluorescence quantitative real-time PCR (qPCR) system (Applied Biosystems, USA) was used in this study.
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!