Samples were lysed in RIPA buffer (P0013B, Beyotime) containing protease inhibitor cocktail (P8340, Sigma) and PMSF (A610425-0005, Sangon Biotech). Proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride membranes. Blocking and antibody incubations were performed in 5% BSA or non-fat dry milk. Anti- PPARγ (16,643-1-AP, 1:1000), anti-FABP4 (12,802-1-AP, 1:1000), anti-PERILIPIN2 (15,294-1-AP), anti-PGC1α (66,369-1-Ig) and anti-UCP1 (23,673-1-AP) were purchased from Proteintech. Anti-β-actin (A5441, 1:1500) was purchased from Sigma-Aldrich. Antibody detection reactions were developed by enhanced chemiluminescence reagent (Millipore) and imaged using the MiniChemi 610 imaging system (Sage Creation). Quantification was done using Lane 1D software (Sage Creation).
Lane 1d software
Manufactured by Sagecreation
Sourced in China
Lane 1D software is a computational tool for analyzing and interpreting one-dimensional electrophoresis gel data. It provides basic functionality for visualizing and quantifying band patterns in gel images.
Automatically generated - may contain errors
Lab products found in correlation
Radio immunoprecipitation assay lysis buffer, by Meilun (2 mentions)
Bca kit, by Beyotime (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Minichemi 610 imaging system, by Sagecreation (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Sangon (1 mentions)
Anti ucp1 23673 1 ap, by Proteintech (1 mentions)
Anti fabp4, by Proteintech (1 mentions)
Anti pparγ 16643 1 ap, by Proteintech (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Skimmed milk, by Biosharp (1 mentions)
Quant qrt pcr kit, by Tiangen Biotech (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Truseq rna library preparation kit, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Human chemokine array c1, by RayBiotech (1 mentions)
Bradford reagent, by Aidlab (1 mentions)
Champchemi professional image analysis system, by Sagecreation (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
6 protocols using lane 1d software
1
Western Blot Analysis of Adipocyte Markers
2
Western Blot Analysis of Murine Intestinal Tissues
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Jejunal and colonal tissues of mice were lysed using the Radio-Immunoprecipitation Assay lysis buffer (MA0151, Dalian Meilun Biotechnology co., Ltd., Dalian, China), centrifuged at 13,680 x g, 4°C, for 30 min, then the supernatant was collected. Protein concentration was measured by the BCA kit (P0011, Beyotime, Shanghai, China). Equal amounts of protein (40 μg) were separated through the SDS-PAGE, subsequently transferred to a PVDF membrane. The PVDF membrane was blocked using the 5% skimmed milk (0040895, Biosharp, Hefei, China) in TBST buffer at room temperature for 1 h, incubated with primary antibodies in 4°C for overnight, and then incubated with HRP (horseradish peroxidase)-labeled secondary antibodies, the signals were detected via the enhanced chemiluminescence reagent. The primary and secondary antibodies were listed in Supplementary Table 3 . The quantification of western blot bands was analyzed using the Lane 1d software (version 5.1.0.0; SageCreation).
3
Quantifying Gene Expression in Embryonic Samples
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Total RNA was reverse transcribed using M-MLV reverse transcriptase (Invitrogen). Semi-quantitative PCR was performed using gene-specific primers, with ß-actin as a loading control (S1 Table ). The intensity of PCR products was analyzed using the Lane 1D software (Sagecreation). Quantitative PCR was performed using Quant qRT-PCR Kit (Tiangen) with gene-specific primers (S1 Table ). RNA sequencing was performed on Illumina HiSeq 2000, using 12 hpf mRNA libraries constructed by TruSeq RNA Library Preparation Kit. The data were aligned and analyzed as described [65 (link)].
4
Human Chemokine Array Evaluation
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Human Chemokine Array C1 from RayBiotech (Atlanta, GA, USA) contains 38 repeated antibodies spotted on a single membrane. Experiments were implemented according to the manufacturer’s protocol, and the results were analyzed by both Lane 1D software from Sage Creation (Beijing, China) and software from RayBiotech.
5
Western Blot Analysis of Protein Extracts
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Cells were washed twice with ice-cold PBS and then lysed in RIPA lysis buffer (CWBIO). The protein concentration of the lysates was determined with the Bradford reagent (Aid Lab). Equal amounts of proteins were separated on SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% fat-free milk and immunoblotted overnight at 4 °C with primary antibodies followed by the corresponding secondary antibodies for 1 h. β-actin was used as the loading control. The membranes were detected using enhanced chemiluminescent (ECL) Prime Western blotting detection reagent (Amersham Biosciences, Uppsala, Sweden). Images were acquired with a Champchemi Professional image analysis system (Sagecreation, Beijing, China) and quantified using LANE 1D software (Sagecreation).
6
Protein Expression Analysis in Liver Tissue
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Liver tissues were lysed in Radio-Immunoprecipitation Assay lysis buffer (MA0151, Dalian Meilun Biotechnology co., Ltd., Dalian, China), and then centrifuged at 13,680 x g, 4°C, 30 min, and then the supernatant was harvested. Protein concentration was measured via BCA kit (P0011, Beyotime, Shanghai, China). After that, equal amounts of protein (30 μg) were separate via the SDS-PAGE, and subsequently transferred to the PVDF membrane. The PVDF membrane was blocked with 5% skimmed milk (0040895, Biosharp, Hefei, China) in TBST buffer for 1 hour at room temperature, then incubated with primary antibodies in 4°C for overnight, and then incubated with HRP (horseradish peroxidase)-labeled secondary antibodies, the signals were detected using enhanced chemiluminescence reagent. The primary antibodies included: rabbit anti-GAPDH (1:2,500; ab9485; Abcam), rabbit anti-CD36 (1:1000; ab133625; Abcam), mouse anti-HNF4a (1:5,000; ab41898; Abcam), rabbit anti-HMGCR (1:1,000; ab174830; Abcam), mouse anti-SCD1 (1:1,000; ab19862; Abcam), rabbit anti-FASN (1:1,000; #3180; CST). Secondary antibodies included HRP-goat anti-rabbit IgG (1:5,000; os0701; Earthox Life Sciences) and HRP-donkey anti-mouse IgG (1:2,000; ab150105; Abcam). The quantification of WB bands was analyzed through the Lane 1d software (version 5.1.0.0; SageCreation).
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