To investigate the expression level of ADAM29 in the MGC803 and AGS cells, confluent cells were centrifuged at 2,400 × g for 10 min at 4°C and lysed with lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The protein concentration was measured with a bicinchoninic acid protein assay kit (Beijing ComWin Biotech Co., Ltd., Beijing, China). A total of 5 µg 20 µl proteins were separated with 10% SDS-PAGE and blotted on to a nitrocellulose membrane. Following protein transfer, the membrane was treated with 5% skimmed milk to block non-specific proteins for 1 h at room temperature. Specific proteins were separately probed with the aforementioned anti-ADAM29 primary antibody (1:1,000; Abnova, Taipei, Taiwan) and anti-GAPDH antibody (1:1,000; cat no. sc-32233; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C, and then followed by incubation with aforementioned peroxidase-conjugated secondary antibody (1:200) for 1 h at room temperature. Protein bands were visualized using the Tanon High-sig ECL (Tanon Science and Technology Co., Ltd., Shanghai, China) and analyzed with Vilber Fusion Fx5 Spectra (Vilber Lourmat, Marne La Vallée, France).
Fusion fx5 spectra
Manufactured by Vilber
Sourced in France, China
The Fusion FX5 Spectra is a multipurpose laboratory instrument designed for various imaging applications. It features a high-resolution camera and advanced imaging capabilities to capture and analyze samples.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Solarbio (2 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (2 mentions)
Bca protein assay, by Applygen (2 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Mtor antibody, by Abcam (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Pi3k antibody, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by ZSGB-BIO (1 mentions)
Akt antibody, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein assay kit, by CWBIO (1 mentions)
β actin, by CWBIO (1 mentions)
Lamin b1, by Huabio (1 mentions)
Slc7a11, by Affinity Biosciences (1 mentions)
P akt, by Proteintech (1 mentions)
P pi3k, by Affinity Biosciences (1 mentions)
Goat anti mouse rabbit igg horseradish peroxidase conjugated antibody, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
S0027, by Beyotime (1 mentions)
9 protocols using fusion fx5 spectra
1
ADAM29 Expression in MGC803 and AGS Cells
2
Western Blot Analysis of AKT/PI3K/mTOR Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were separated with 10% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, MA). Following being blocked with 5% skimmed milk to block non-specific proteins for 1 h at room temperature, membranes were treated with corresponding primary antibody overnight at 4°C, and then followed by peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies for 1 h at room temperature. Protein bands were visualized using the Tanon High-sig ECL (Tanon Science and Technology Co., Ltd., Shanghai, China) and analyzed with Vilber Fusion Fx5 Spectra (Vilber Lourmat, Marne La Vallée, France). The anti-DEPDC1 antibody was purchased from abcam Inc. (Abcam, Cambridge, MA, USA). GAPDH antibody was obtained from Santa Inc (Dallas, TX, USA). AKT antibody (Cell Signaling Technology, USA), p-AKT (Ser473) (Cell Signaling Technology, USA), PI3K antibody (Santa Cruz, USA), p-PI3K p85 (Tyr458) antibody (Santa Cruz, USA), mTOR antibody (Abcam, USA) and p-mTOR (Ser2448) antibody (Cell Signaling Technology, USA) were used to analyze AKT/PI3K/mTOR signal pathway. HRP-conjugated secondary antibodies were from ZSGB-BIO (Beijing, China).
3
Protein Expression Analysis in Renal Cortex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Renal cortex tissue samples were homogenized in RIPA lysis buffer, and the Bicinchoninic Acid Protein Assay Kit (Cwbiotech, Beijing, China) was used to measure protein levels, as previously described (Guan et al., 2019 (link)). 40 μg protein from renal homogenate was applied to each lane for separation on SDS–PAGE. Immunoblotting was performed on the separated protein and probe with the following primary antibodies overnight at 4°C: TfR1 (1:2,000, Cat#abs131442, absin), FTL (1:10,000, Cat#3549-1, EPITMICS), DMT1 (1:2,000, Cat#abs112967, absin), FPN1 (1:3,000, Cat#GTX100573, absin), NOX2 (1:2,000, Cat#BA2811, BOSTER), NOX4 (1:1,000, Cat#GB11347, servicebio), GPx4 (1:2,000, Cat#ET1706-45, HUABIO), SLC7A11 (1:1,000, Cat#DF12509, Affinity), Nrf2 (1:1,000, Cat#M200-3, MBL), HO-1 (1:2,000, Cat#GB12104, servicebio), p-Pi3K (Cat#AF3241, Affinity), Pi3K (Cat#AF6241, Affinity), p-Akt (Cat#66444-1-Ig, Proteintech), Akt (Cat#60203-2-Ig, Proteintech), β-actin (1:1,000, Cat#CW0096S, CWBIO), and Lamin B1 (1:1,500, Cat#SI17-06, HUABIO). The secondary antibodies were diluted at 1:1,000. The blots were imaged and analyzed applying enhanced chemiluminescent detection (Vilber Fusion FX5 Spectra, Paris, France). The proteins were normalized against β-actin.
4
Western Blot Analysis of Prostate Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prostate cancer cells (1×106) cultured in 100 mm2 culture dishes were treated with various concentrations of PD for
24 hr. Whole cell lysates were obtained by cell lysis in ice-cold RIPA buffer and subjected to SDS-PAGE according to a previously reported procedure [17 (link)]. The PVDF membrane was incubated with the appropriate primary antibody overnight at 4 °C with gentle shaking. The membrane was then incubated with a goat anti-mouse/rabbit IgG horseradish peroxidase-conjugated antibody (Bio-Rad). Conjugated proteins were detected by the Fusion FX5 Spectra instrument from VilberLourmat Inc. (France).
5
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
According to the previously described method [19 (link)], total protein extraction was applied with RIPA buffer, adding a protease inhibitor cocktail, phenylmethylsulfonyl fluoride, and phosphatase inhibitors. Protein concentration was measured using a bicinchoninic acid protein assay kit (Wuhan Servicebio Technology, Ltd., China). Equal amounts of protein were loaded into the wells of SDS-PAGE (10%) and separated. A semidry transfer printer (Bio-rad, United States) was used to transfer protein from gel to PVDF membranes. Nonfat milk (5%) was used to block nonspecific epitopes for 90 min. The membranes were incubated with primary antibodies for α-SMA, TGF-β1, Collagen I, Collagen III, SOX9, Smad3, phospho-Smad3, and GAPDH according to instructions overnight at 4°C. After washing three times with TBST, the membranes were incubated by the corresponding secondary antibodies labelled with HRP for 90 min at RT. The blot bands were visualised by the ECL (Invitrogen) reagents and Fusion FX5 Spectra (VilberLourmat, France). Each protein was quantified by transmittance densitometry using Image J 3.0 software (National Institute of Health).
6
Mitochondrial ATP Quantification in Ovarian Granulosa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial ATP content was detected using an ATP detection kit (S0027, Beyotime Institute of Biotechnology, China). A total of 200 μl of lysate was added to each tube of ovarian GCs sample for centrifugation at 12,000g for 5 min. The supernatant was removed for future use. The standard solution and test working solution were prepared according to the instructions. Then, the sample supernatant, standard solution, and test working solution were added to a 96-well plate according to the instructions. The readings were made using Fusion FX5 Spectra (Vilber Lourmat, Marne-la-Vallée, France), and the concentrations were calculated according to the standard curve concentration. To boost accuracy, the protein concentrations of each group of samples were determined by a BCA protein concentration assay kit (PC0020, Solarbio, China), and the ATP content was converted to μmol/mg protein.
7
Western Blot Analysis of TLR4 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The heart tissues were homogenized, and the lysates were collected to obtain the total protein using lysis buffer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). Nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology Co., Ltd., Beijing, China), according to the manufacturer’s protocol. Total protein and nuclear protein were extracted and their concentrations in all samples were quantified with the bicinchoninic acid (BCA) protein assay (Applygen Technologies Inc., Beijing, China). Subsequently, proteins were separated on 10% SDS-PAGE gel and transferred to a PVDF membrane. After blocking in 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies against TLR4 (1:1000), NF-κB (1:1000), IL-6 (1:1000), TAK-1 (1:750), GAPDH (1:10000) and H3 (1:10000). Then the membranes were incubated with an HRP-conjugated secondary antibody followed by ECL detection (Vilber Fusion FX5 Spectra, Paris, France). And the bands were analyzed semi-quantitatively with Image J software (National Institutes of Health, Bethesda, Maryland, USA).
8
Western Blot Analysis of OPD' Exposure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (1 × 106) cultured in 25 mm2 culture flasks were exposed to various concentrations of OPD′ for 6 h. Whole cell lysates were obtained by cell lysis in ice-cold RIPA buffer and were subjected to SDS-PAGE, according to a previously reported procedure (Lu et al., 2016 (link)). PVDF membranes with the adherent proteins were incubated with the selected primary antibody overnight at 4°C with gentle shaking. The membrane was then incubated with a goat anti-mouse/rabbit IgG horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA, United States). Conjugated proteins were detected by the Fusion FX5 Spectra instrument from Vilber Lourmat, Inc. (Marne-la-Vallée, France).
9
Protein Expression Analysis in Heart Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The heart tissues were homogenized and the lysates were collected to obtain the total protein using lysis buffer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China).
Nuclear proteins were extracted using nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology Co., Ltd., Beijing, China), according to the manufacturer's protocol. Total protein and nuclear protein were extracted and their concentrations in all samples were quantified with the bicinchoninic acid (BCA) protein assay (Applygen Technologies Inc., Beijing, China). Subsequently, proteins were separated on 10% SDS-PAGE gel and transferred to a PVDF membrane. After blocking in 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies against TLR4 (1:1000), NF-κB (1:1000), IL-6 (1:1000), TAK-1 (1:750), GAPDH (1:10000), and H3 (1:10000). Then the membranes were incubated with an HRP-conjugated secondary antibody followed by ECL detection (Vilber Fusion FX5 Spectra, Paris, France). And the bands were analyzed semi-quantitatively with Image J software (National Institutes of Health, Bethesda, Maryland, USA).
Nuclear proteins were extracted using nuclear and cytoplasmic protein extraction kit (Beyotime Biotechnology Co., Ltd., Beijing, China), according to the manufacturer's protocol. Total protein and nuclear protein were extracted and their concentrations in all samples were quantified with the bicinchoninic acid (BCA) protein assay (Applygen Technologies Inc., Beijing, China). Subsequently, proteins were separated on 10% SDS-PAGE gel and transferred to a PVDF membrane. After blocking in 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies against TLR4 (1:1000), NF-κB (1:1000), IL-6 (1:1000), TAK-1 (1:750), GAPDH (1:10000), and H3 (1:10000). Then the membranes were incubated with an HRP-conjugated secondary antibody followed by ECL detection (Vilber Fusion FX5 Spectra, Paris, France). And the bands were analyzed semi-quantitatively with Image J software (National Institutes of Health, Bethesda, Maryland, USA).
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!