Cells in the logarithmic growth phase were lysed for 30 minutes with RIPA lysis buffer, and then centrifuged at 12,000 g for 5 mins. Protein quantification was performed using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL). 10 ug protein was used for SDS-PAGE electrophoresis. Separated protein in SDS-PAGE gel was transferred onto the PVDF membrane (BioRad 1620177, Irvine, CA, USA). After blocking with 5% skimmed milk for 1 hour, the membrane was then incubated with primary antibodies: rabbit mAb CD63 (1:1,000; Abcam), rabbit mAb Hsp70 (1:1,000; StressGen), rabbit mAb HMGA1 (1:100; Abcam), and rabbit pAb -actin (1:1,000, Cell Signaling Technology). The primary antibodies were dissolved in TBST comprising 5% BSA and incubated overnight at 4°C. The membrane was washed 3 times with TBST for 5 minutes each. After wash, the membrane was further incubated with goat anti-rabbit secondary antibody conjugated with HRP (1:3000; Cell signaling #7074, MA, USA). Then the membrane was washed 4 times with TBST and the protein bands were visualized using an EZ-ECL Chemiluminescence Detection kit (Pierce, Rockford, IL) and photographed on a gel imager system (Bio-Rad, Hercules, CA, United States). The densitometry analysis was performed with Image J software (Bethesda, MD, USA).
Gel imager system
Manufactured by Bio-Rad
Sourced in United States, China
The Gel Imager System is a laboratory equipment designed for capturing and analyzing images of electrophoresis gels. It provides a consistent and reliable platform for visualizing and documenting various types of stained gels, such as those used in DNA, protein, and other biomolecular analyses.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (20 mentions)
Enhanced chemiluminescence kit, by Santa Cruz Biotechnology (14 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (14 mentions)
Bca protein assay kit, by Beyotime (14 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Bca protein assay kit, by Solarbio (4 mentions)
Ab9485, by Abcam (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Hmga1, by Abcam (1 mentions)
Hsp70, by Stressgen (1 mentions)
Ab236639, by Abcam (1 mentions)
Ab133612, by Abcam (1 mentions)
37 protocols using gel imager system
1
Western Blot Analysis of Protein Expression
2
Genotyping of human TP53 polymorphism
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Genotyping of human Tp53 polymorphism was performed at the University of Salamanca by authors blinded to the clinical status of patients, by using the PCR-RFLP technique.23 (link) The Tp53 SNP of exon 4 at codon 72 (Arg72Pro; rs1042522) was detected by amplifying genomic DNA with the forward primer 5′-TCTACAGTCCCCCTTGCCGT-3′ and the reverse primer 5′-CTGACCGTGCAAGTCACAGA-3′. Tp53 exon 4, where BstU1 (Bsh1236I) RFLP is located, was amplified within a 298-bp DNA fragment. Digests were separated on agarose gel (3%) and the Midori Green Advanced-stained DNA fragments (Nippon Genetics Europe GmbH, Düren, Germany) were analyzed under a UV source (Gel Imager System; Bio-Rad, Hercules, CA, USA). The distribution of genotype frequencies in Arg72Pro between the ICH cohort was within the Hardy–Weinberg equilibrium (P>0.1 in all cases). As the Pro allele is likely to exert a dominant effect on the Arg one,23 (link) two groups were considered for this study: Pro (Pro/Pro and Pro/Arg genotypes) and Arg (Arg/Arg genotype) patients.
3
Quantitative Protein Analysis of Osteogenic Markers
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Total proteins were extracted using RIPA reagent (Beyotime Biotechnology, Shanghai, China) and the protein concertation was determined by BCA kit (Beyotime Biotechnology, Shanghai, China) as per the instructions. Then, 25 μg of protein was loaded on 10% SDS-PAGE and transferred on PVDF membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk for 1 h, PVDF membranes were incubated with primary antibodies (rabbit anti-RUNX2, 1:1000, ab236639; rabbit anti-OCN, 1:1000, ab133612; rabbit anti-OSX, 1:1000, ab209484; rabbit anti-OPN, 1:1000, ab8448; rabbit anti-GAPDH, 1:1000, ab9485; all purchased from Abcam, Shanghai, China) overnight at 4°C. The following day, membranes were washed 3 times with TBST buffer for 5 minutes each, and further incubated with secondary antibody (goat anti-rabbit H&L preadsorbed, 1:1000, ab7090; purchased from Abcam, Shanghai, China) for 2 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence kit (Yeasen, Shanghai, China) and photographed on a gel imager system (Bio-Rad, Hercules, CA, United States). GAPDH functioned as the internal control.
4
Plasmid Size Determination by PFGE
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Pulsed-field gel electrophoresis (PFGE) with S1 nuclease (Takara Biotechnology, Dalian, China) digestion was carried out to determine the size of the plasmid. Briefly, after cells (OD600 = 0.95 − 1.00) were fixed with SeaKem Gold agarose (Cambrex BioScience, Walkersville, MD, USA) and subsequently lysed, the embedded DNAs were digested using 18 U S1 enzymes (Takara Biotechnology, Dalian, China) in a 37 °C water bath for 15 min. The restricted DNA fragments were separated in 0.5 × TAE buffer at 14 °C for 19 h using a CHEF Mapper electrophoresis system (Bio-Rad, Richmond, CA, USA) with pulse times of 2.16–63.8 s. The PFGE image was obtained with a Gel Imager System (Bio-Rad, USA). S. Braenderup H9812 was used as the DNA size marker.
5
Protein Extraction and Western Blot Analysis
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Total protein was extracted from NSCLC tumor tissues and cells using RIPA lysis buffer containing protease inhibitor cocktail (Invitrogen, USA), then quantified by a BCA Protein assay kit (Solarbio, Beijing, China). 10 ug protein was used for SDS-PAGE electrophoresis. Separated protein in SDS_PAGE gel was transferred onto the PVDF membrane (BioRed, USA). After blocking with 5% skimmed milk for 1 h, the membrane was then incubated with primary antibodies: FGF11 (1:1000; Cell Signaling Technologies #3139, MA, USA), HIF-1α (1:1000, Cell Signaling Technologies #3716), β-Actin (13E5) (1:2000, Cell Signaling Technologies #4970) and GAPDH (1:2000; Cell Signaling Technologies #2118) for 2 h or overnight at 4℃. The membrane was washed 3 times with TBST for 5 min each. After wash, the membrane was further incubated with HRP-linked secondary antibody (1:3000; Cell signaling #7074, MA, USA) at room temperature for 1 h. Then the membrane was washed 4 times with 1 × TBST and the protein bands were visualized using an enhanced chemiluminescence kit (Santa Cruz, TX, USA) and photographed on a gel imager system (Bio-Rad).
6
Protein Quantification and Western Blot Analysis
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RIPA lysis buffer containing protease inhibitor cocktail (Thermo Fisher Scientific) was used for protein sample extraction. The supernatant containing total protein lysate was quantified by a BCA Protein assay kit (Beyotime). 10 μg of protein sample was separated on a SDS‐PAGE gel and then transferred to PVDF membrane. After blocking with 5% skimmed milk, the membrane was incubated with primary antibodies at 4°C overnight: E2F3 (1: 2000, ab152126, Abcam), GAPDH (1: 5000, ab8245, Abcam). The membrane was further incubated with HRP‐linked secondary antibody (1:3000, ab97023, Abcam) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence kit and photographed on a gel imager system (Bio‐Rad).
7
Quantification of PprI Protein Expression
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To investigate the level of PprI expression in L. lactis, strains MG(PprI+) and MG(Vector) were grown in GM17 medium containing 500 mg/mL erythromycin at 30°C. Cell cultures at OD600 1.0 were harvested by centrifugation at 4°C for 10 min. The cells were washed twice with ice-cold PBS buffer, resuspended in the same buffer and disrupted ultrasonically at 4°C for 99 cycles of 3 s each. The cell extract was obtained after centrifugation at 4°C for 10 min to remove cellular debris. The protein concentration was determined according to the Bradford method using bovine serum albumin as the standard. For protein analysis, the cell extract was mixed with a five-fold concentrated buffer; after being heated at 100°C for 10 min, a 15-μL aliquot of each sample was subjected to 12% SDS-PAGE. The proteins in the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (Amersham Pharmacia Biotech, Buckinghamshire, England) and incubated with a rabbit anti-PprI polyclonal antibody (rabbit IgG, laboratory stock). Chemiluminescent signals on the PVDF membrane were visualized and quantified using a Gel Imager System (Bio-Rad Laboratories, Hercules, CA, USA) [12 (link)].
8
Serum Stability of FA/PS/miR-34a/PS@NDs
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The serum stability of FA/PS/miR-34a/PS@NDs were analyzed by 2% agarose gel electrophoresis. The gels were freshly prepared with 2% agarose in tris–acetate–ethylenediaminetetraacetic acid (EDTA) buffer containing 0.5 μg ml−1 GelREDTM (Biotium). The FA/PS/miR-34a/PS@NDs were mixed with FBS at serum concentration of 50%. After the incubation for the indicated times at 37 °C, the same amount (15 μl) of each sample were loaded onto agarose gel for electrophoresis at 110 V for 10 min and imaged using gel imager system (Bio-rad).
9
Quantitative Protein Extraction and Analysis
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Total proteins were extracted from cells and tissues by RIPA lysis and extraction buffer (Thermo Fisher, USA) as previously described [5 (link)]. The supernatant containing total protein lysate was quantified by a BCA Protein assay kit (Beyotime, Shanghai, China). 10 ug of total protein was used for SDS-PAGE electrophoresis and then transferred onto the PVDF membrane. After blocking with 5% skimmed milk for 1 hour, the membrane was then incubated with primary antibodies (Cell Signaling Technology, USA) overnight at 4°C. The membrane was washed 3 times with TBST and incubated with HRP-linked secondary antibody (Cell Signaling Technology, USA) at room temperature for 1 hour. Protein bands were developed using an enhanced chemiluminescence kit (Santa Cruz, TX, USA) and photographed on a gel imager system (Bio-Rad, CA, USA). The densitometry analysis was performed with Image J software (Bethesda, MD, USA).
10
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells using RIPA lysis buffer containing protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Cells suspended in RIPA buffer were lysed on ice for 10 mins and lysed cells were centrifuged at 14,000 rpm for 10 mins. The supernatant containing total protein lysate was quantified by a BCA Protein assay kit (Beyotime Biotechnology P0009; Shanghai, China). 20 µg of protein was loaded into 12% sodium dodecyl sulfate salt-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk the membrane was incubated with primary antibody IRS2 (1:5000, Abcam) overnight at 4°C. The membrane was washed 3 times with TBST for 5 minutes each. After wash, the membrane was further incubated with HRP-linked secondary antibody (1:2500; Cell Signaling Technologies, MA, USA) at room temperature for 1 hour. Then the membrane was washed 4 times with TBST and the protein bands were visualized using an Electro-Chemi-Luminescence kit (Santa Cruz, TX, USA) and photographed on a gel imager system (Bio-Rad, Hercules, CA, United States). The densitometry analysis was performed with Image J software (Bethesda, MD, USA) [20 (link)].
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