Tissue or cells in logarithmic growth phase were added to the cell lysate to extract protein. The protein concentration was determined by BCA method (Sigma). The uploading volume of the sample was adjusted according to the protein quantification result. 30 μg was loaded per well. The protein was separated by SDS-PAGE and transferred onto the PVDF membrane. The membrane was blocked by 50 g/L skim milk powder for 1 h, followed by TBST solution washing for 3 times. Then the samples were incubated with primary antibodies rabbit anti-human PTEN (1:1000, Cat No. 32199, Abcam, Cambridge, UK), Beclin1 (1:1000, Cat No. 62557, Abcam), LC-3 (1:3000, Cat No. 51520, Abcam), hTERT (1:1000, Cat No. 32020, Abcam), and β-actin (1:5000, Cat No. 8227, Abcam) overnight at 4°C on a shaker. After three washes with TBST, HRP-labelled goat anti-rabbit IgG antibody (1:3000 dilution, Cat no. 205718, Abcam, Cambridge, UK) was added for incubation for 1 h, followed by TBST wash for 3 times. The target band was detected by ECL chemiluminescence and recorded using the Bio-image system (Bio-Rad, Hercules, CA, USA). The level of protein expression was normalized with β-actin as an internal reference.
Bio image system
Manufactured by Bio-Rad
Sourced in United States
The Bio-image System is a laboratory instrument designed for imaging and analyzing biological samples. It provides high-resolution digital imaging capabilities for a variety of applications, such as gel electrophoresis, Western blotting, and other imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Htert, by Abcam (1 mentions)
Hrp labelled goat anti rabbit igg antibody, by Abcam (1 mentions)
Bca method, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Beclin 1, by Abcam (1 mentions)
Dcode universal mutation detection system, by Bio-Rad (1 mentions)
Model 475, by Bio-Rad (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Ab81046, by Abcam (1 mentions)
Ab189073, by Abcam (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by ABclonal (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Apaf 1, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Sds lysis buffer, by Beyotime (1 mentions)
Dnagreen uv, by Tiandz (1 mentions)
7 protocols using bio image system
1
Western Blot Analysis of Autophagy Proteins
2
Denaturing Gradient Gel Electrophoresis Analysis
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DGGE was performed using a Dcode universal mutation detection system (Bio-Rad Inc., Hercules, CA, USA). Samples containing approximately equal amounts of PCR amplification products were loaded on a 10% poly-acrylamide gel in TAE buffer with a denaturing gradient ranging from 30% to 60% of the denaturant (denaturation of 100% corresponded to 7 mol·L−1 urea and 40% (v/v) formamide). DGGE was performed at 60 °C with a constant voltage of 160 V for 6 h, by using a gradient delivery system (Model 475, Bio-Rad Inc., Hercules, CA, USA. The gel was then stained by AgNO3 and the images of the gel were captured by a bio-image system (Bio-Rad).
DGGE profiles were analyzed using the software Quantity One 4.6.2 (Bio-Rad Inc., Hercules, CA, USA). Dendrograms relating band pattern similarities were automatically calculated using the unweighted pair group method with the arithmetic average (UPGMA) clustering algorithm. UPGMA employs a sequential clustering algorithm, in which local topological relationships are identified in order of similarity, and the phylogenetic tree is built in a stepwise manner. Relative band densities, which were necessary to determine the Shannon diversity index (SDI), were quantified, and the statistical data were exported for further SDI analyses.
DGGE profiles were analyzed using the software Quantity One 4.6.2 (Bio-Rad Inc., Hercules, CA, USA). Dendrograms relating band pattern similarities were automatically calculated using the unweighted pair group method with the arithmetic average (UPGMA) clustering algorithm. UPGMA employs a sequential clustering algorithm, in which local topological relationships are identified in order of similarity, and the phylogenetic tree is built in a stepwise manner. Relative band densities, which were necessary to determine the Shannon diversity index (SDI), were quantified, and the statistical data were exported for further SDI analyses.
3
Quantification of LIMK1 and Phospho-LIMK1
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Isolated left hippocampus tissue was lysed in 100 µL radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Haimen, Jiangsu, China) plus protease inhibitors. Total protein (50 µg) was loaded into 10% SDS-PAGE gels, electrophoresed, and then transblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA) in a Tris-glycine transfer buffer. After being blocked in 5% milk in PBST for 1 h at room temperature with shaking, membranes were incubated with antibodies overnight at 4°C against the following: anti-LIMK1 antibody (dilution, 1 : 1000; ab81046, Abcam) and anti-LIM kinase 1 antibody (phospho-Thr508) (dilution, 1 : 1000; ab131341, Abcam) and β-actin (dilution, 1 : 1000; ab189073; Abcam). The following day, membranes were incubated in 5% milk (in TBST) with an anti-goat or anti-mouse IgG antibody (dilution, 1 : 5000; PerkinElmer Life Sciences, Waltham, MA) for 1 h at room temperature with shaking. Membranes were washed a minimum of four times (10 min per wash) in PBST between each antibody treatment. Detected bands were visualized using enhanced chemiluminescence and images were captured using a Bio-Image system (Bio-Rad Laboratories, Inc., Hercules, USA). Western blotting was repeated three times.
4
Protein Extraction and Western Blot Analysis of Renal Tissues
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The renal tissues were lysed in sodium dodecyl sulfate (SDS) lysis buffer (Beyotime, China) for extracting total protein, and the concentration of protein was determined using the bicinchoninic acid protein assay kit (Thermo Fisher, USA). Then, 40 µg total protein was loaded into each lane and separated by SDS-polyacrylamide electrophoresis. The proteins in the SDS gel were transferred onto a polyvinylidene difluoride membrane (Millipore, USA), and the nonspecific binding protein was blocked in Tris-buffered saline with Tween 20 (TBST) buffer containing 5% skimmed milk for 1 h at 37°C. The membrane was incubated using primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000; Abcam), FADD (1:100; Abcam), Apaf-1 (1:2000; Abcam), and CHOP (1:1000; Abcam) overnight at 4°C on a shaking table. Sequentially, the membrane was immersed in goat anti-rat immunoglobulin G (IgG; 1:5000; ABclonal, USA) or goat anti-rabbit IgG (1:5000; ABclonal) antibody for 1 h at room temperature. The membrane was washed three times using TBST for 10 min after each step. The protein bands were visualized using an enhanced chemiluminescence system detection kit (ECL; Amersham, USA) in a bioimage system (Bio-Rad, USA). The grayscale value was calculated using Image-Pro Plus 6.0 software. Five samples were selected randomly for western blot analysis.
5
Evaluating TGF-β Expression in 4T1 Tumor Model
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As mentioned before, 4T1 tumor-bearing mice were injected with 5% Glu, Fu (50 mg/kg), DOX, DD/DOX or FD/DOX (DOX-equivalent 2.5 mg/kg) through tail vein, respectively, every 3 days for a total of 7 times. After the treatment, tumors, lungs and livers from mice were collected, and the sections of each tissue were applied for TGF-β staining. The TGF-β was also detected by Western blot assay. Tumors, lungs and livers from mice were collected and homogenized to collect proteins. The obtained proteins were first separated by acrylamide gel (12% SDS-PAGE). Next, the separated proteins were transferred to a polyvinylidene difluoride membrane. After that, the membrane was incubated with anti-TGF-β antibody and the secondary antibody in sequence. Finally, a Bioimage System (Bio-Rad Laboratories Inc., ChemiDoc MP, CA, USA) was used to measure the expression of TGF-β.
6
RAPD Analysis of Genetic Diversity
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The RAPD analysis was performed using nine primers as previously described by Powell et al. [37] (link). The composition of the PCR reaction mixture contained 2.2 µL of DNA template, 2 µL 10×PCR Buffer, 1.5 µL dNTPs, 0.2 µL Taq polymerase, 0.5 µL Primer, and 13.6 µL ddH2O. The cycling conditions were as follows: 4 min at 94 • C for an initiation step, followed by 40 cycles of 30 s at 94 • C, 30 s at a primer-appropriate temperature, and 2 min at 72 • C, and a final cycle of 10 min at 72 • C. Amplified products were separated on 2% agarose gel in 1× TAE buffer by electrophoresis at 100 V for 40 min and photographed by Bio-image System (BioRad, CA, USA).
7
ISSR-PCR Amplification Protocol
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A total of 100 ISSR primers (University of British Columbia, Vancouver, BC, Canada) were tested with nine (UBC #808, #824, #827, #836, #841, #842, #847, #857, and #873) showing high reproducibility and accuracy selected for PCR amplification. The PCR reaction was performed as described in literature with a minor modification [36] (link). The optimal reaction condition of ISSR-PCR amplification contained 2.5 µL template DNA, 2 µL 10× PCR buffer, 1.5 µL dNTPs, 0.2 µL Taq polymerase, 0.5 µL primer, 13.3 µL ddH 2 O to make the total volume of 20 µL.
The cycling conditions were as follows: 4 min at 94 • C for an initiation step, followed by 40 cycles of 30 s at 94 • C, 30 s at a primer-appropriate temperature, and 2 min at 72 • C, and finalized at a final cycle of 10 min at 72 • C. PCR products were detected by electrophoresis on a 2.0% agarose gel stained with 0.1 µL/mL of DNAgreen (UV) (Tiandz, China) and run in 1× TAE buffer at 100 V for 40 min. PCR products were photographed by the Bio-image System (BioRad, Germany). Each PCR reaction was repeated three times.
The cycling conditions were as follows: 4 min at 94 • C for an initiation step, followed by 40 cycles of 30 s at 94 • C, 30 s at a primer-appropriate temperature, and 2 min at 72 • C, and finalized at a final cycle of 10 min at 72 • C. PCR products were detected by electrophoresis on a 2.0% agarose gel stained with 0.1 µL/mL of DNAgreen (UV) (Tiandz, China) and run in 1× TAE buffer at 100 V for 40 min. PCR products were photographed by the Bio-image System (BioRad, Germany). Each PCR reaction was repeated three times.
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