On the basis of previously established protocol of Rong et al,36 proteins were extracted from ileum, eWAT, and liver by using lysing buffer (Solarbio). Roughly 30 μg protein was separated by electrophoresis (12 % and 5 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels) and then transferred onto polyvinylidene difluoride nitrocellulose membranes (Millipore, Burlington, MA), which were further blocked by 5 % non-fat milk in Tris-Tween buffered saline buffer for 2 hours. After blocking, these membranes were incubated with various primary antibodies, including anti-Niemann-Pick C1-Like 1 (NPC1L1), anti-lysophosphatidylcholine acyltransferase 3 (LPCAT3), anti-fatty acid transport protein4 (FATP4), anti-LPL, anti-adipose triglyceride lipase (ATGL), anti-acetyl CoA-carboxylase (ACC), anti-IL-22, anti-STAT3, anti-phosphorylated STAT3 (p-STAT3), anti-ANGPTL4, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, UK), and horseradish peroxidase-conjugated secondary antibody. Finally, proteins were imaged using chemiluminescent peroxidase substrate (Millipore) and quantified using ChemiDoc XRS system (Bio-Rad, Richmond, CA).
Lysing buffer
Manufactured by Solarbio
Sourced in China
Lysing buffer is a solution used to disrupt cell membranes and release cellular contents, including DNA, RNA, and proteins, for further analysis or processing. It is a core component in various molecular biology and biochemistry procedures.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride nitrocellulose membranes, by Merck Group (1 mentions)
Chemiluminescent peroxidase substrate, by Merck Group (1 mentions)
Anti angptl4, by Abcam (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ter119, by BioLegend (1 mentions)
Lineage cocktail, by BioLegend (1 mentions)
Sca 1, by BioLegend (1 mentions)
Annexin 5 staining kit, by BD (1 mentions)
Cd11b, by BD (1 mentions)
2 protocols using lysing buffer
1
Ileum, eWAT, and Liver Protein Analysis
2
Murine Hematopoietic Lineage Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow cells were flushed from the femurs and tibias of mice with PBS containing 2% FBS and lysed with lysing buffer (catalogue number: R1010, Solarbio, China) for red blood cell deletion. Bone marrow cells were suspended in PBS and stained by antibodies for 30 min. For Detection lineage-specific markers: Erythroid: Ter119 (FITC, Biolegend, catalogue number: 116206) and CD71 (PE, eBioscience, catalogue number:12–0711-82), Granular line: CD11b (FITC, BD, catalogue number:557396) and Gr-1 (PE, BD, catalogue number: 553126), Megakaryocyte: CD41 (FITC, BD, catalogue number:553848) and C-kit (PE, BD, catalogue number:553355), LSK: lineage cocktail (FITC), sca-1 (APC, Biolegend, catalogue number:108112) and C-kit (PE, BD, catalogue number:553355). Cell apoptosis analysis was performed using Annexin V staining kit according to the instructions of the manufacturer (BD).
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!