M-PER mammalian protein extraction reagent (Thermo Fisher), containing a protease inhibitor cocktail, was used to extract protein lysate from transfected cells (Thermo Fisher). SDS-PAGE was used to separate 40 μg of protein lysate, followed by blotting onto a nitrocellulose membrane (Bio-Rad, USA). The membrane was blocked using 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. Subsequently, blot was incubated overnight at 4 °C with primary antibodies against GLUT2 (Anti-rabbit; 1:1000, #ab54460, Abcam, UK), INSRβ (Antimouse; 1:1000; #ab69508, Abcam, UK), INSRα (Anti-rabbit; 1:1000; #ab5500, Abcam, UK), Insulin (Anti-mouse; 1:1000; #8138s, Cell signaling Technology, USA), GCK (Anti-rabbit; 1:500; #ab37796, Abcam, UK) PDX1 (Anti-rabbit; 1:3000, #ab47267, Abcam, UK), or β-actin (1:5000; #A5441, Sigma-Aldrich, Germany). The membrane was incubated with secondary antibodies (anti-mouse #7076S and antirabbit #7074S, Cell signaling Technology, USA) at 1:1000 dilutions at room temperature for 1 h. ECL substrate kit (Bio-Rad, USA) was used to detect chemiluminescence. Bio-Rad Image Lab software (Bio-Rad, USA) was used to detect protein bands. Quantification of the bands was done using Image J software. In all experiments, β-actin was used as an endogenous control (see Supplementary Information ).
Ab5500
Manufactured by Abcam
Sourced in United Kingdom, United States
The Ab5500 is a high-quality laboratory equipment product offered by Abcam. It is designed for use in various scientific and research applications. The core function of this product is to provide accurate and reliable measurements or analysis, as per its intended purpose. However, a more detailed description cannot be provided while maintaining an unbiased and purely factual approach, as requested.
Automatically generated - may contain errors
Lab products found in correlation
Ab32391, by Abcam (2 mentions)
Ab75814, by Abcam (2 mentions)
Ab201060, by Abcam (2 mentions)
Ab134101, by Abcam (2 mentions)
Ab69508, by Abcam (1 mentions)
Ab37796, by Abcam (1 mentions)
Ab47267, by Abcam (1 mentions)
Anti mouse 7076, by Cell Signaling Technology (1 mentions)
Anti rabbit 7074, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ab54460, by Abcam (1 mentions)
A5441, by Merck Group (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ecl substrate kit, by Bio-Rad (1 mentions)
Enhanced chemiluminescence system, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab8226, by Abcam (1 mentions)
Bicinchoninic acid assay kit, by Beyotime (1 mentions)
Ab46800, by Abcam (1 mentions)
6 protocols using ab5500
1
Protein Expression Analysis in Transfected Cells
2
Protein Expression Analysis in Pancreas and Ovary
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was collected from the pancreas and ovary using a radioimmunoprecipitation lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China), and the protein concentration was determined using a bicinchoninic acid assay kit (Beyotime Biotechnology). Equal amounts of proteins (50 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 1 h in 5% skim milk at room temperature and then incubated with primary and secondary antibodies. The following antibodies were used: rabbit anti-INSR (1:200; ab5500, Abcam, Cambridge, UK), rabbit anti-FSHR (1:200; GXP193389, GXP, USA), rabbit anti-LHCGR (1:200; GXP298459, GXP, USA), and mouse anti-β-actin (1:5,000, ab8226, Abcam) primary antibodies with which the membranes were incubated overnight at 4°C, and horseradish peroxidase-conjugated secondary anti-mouse/rabbit antibody for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence system (Millipore) and analyzed with ImageJ 2x (National Institutes of Health, Bethesda, MD, USA). Protein levels were normalized to the respective level of the β-actin internal controls.
3
Protein Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues or cells were lysed in ice-cold radio-immunoprecipitation assay (P0013B, RIPA) buffer containing protease and phosphatase inhibitors (phenylmethylsulfonyl fluoride, ST506, Beyotime, Shanghai, China). Total protein was measured using the bicinchoninic acid protein assay (P0010, BCA, Beyotime, Shanghai, China). Western blot analysis was performed as previously described23 (link), and the antibodies were applied at concentrations according to the manufacturer’s instructions. Immune complexes were detected using the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0050, Millipore Corporation, Billerica, MA). Actin served as the loading control. Bands were quantified using Image Lab Version 2.0.1 (Bio-Rad, Hercules, CA).
Anti-Actin (AA128, Beyotime), anti-GPR109a (ab81825, ABCAm, USA), anti-GPR43 (DF2746, Affinity Biosciences, USA), anti-H3 (anti-total histone H3, 4499, Cell Signaling Technology, CST, USA), anti-Ace-H3 (anti-pan-acetyl histone H3, 61637, Active Motif, USA), anti-p-AMPK (2535, adenosine monophosphate activated protein kinase, CST, USA), anti-p-ACC (11818, p-acetyl-CoA carboxylase, CST), anti-IR (anti-insulin receptor, ab5500, ABCAm), anti-IRS-1 (anti-insulin receptor substrate-1, ab46800, ABCAm), and anti-GLP-1R (NBP1-97308, Novus Biologicals, USA) were used in our study.
Anti-Actin (AA128, Beyotime), anti-GPR109a (ab81825, ABCAm, USA), anti-GPR43 (DF2746, Affinity Biosciences, USA), anti-H3 (anti-total histone H3, 4499, Cell Signaling Technology, CST, USA), anti-Ace-H3 (anti-pan-acetyl histone H3, 61637, Active Motif, USA), anti-p-AMPK (2535, adenosine monophosphate activated protein kinase, CST, USA), anti-p-ACC (11818, p-acetyl-CoA carboxylase, CST), anti-IR (anti-insulin receptor, ab5500, ABCAm), anti-IRS-1 (anti-insulin receptor substrate-1, ab46800, ABCAm), and anti-GLP-1R (NBP1-97308, Novus Biologicals, USA) were used in our study.
4
Immunohistochemical Analysis of Alzheimer's Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The brain tissues of 3 mice from each group were collected and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. The oven was heated to 56 °C, and the selected sections were baked for 1 h. The brain sections were deparaffinized in xylene and rehydrated. For staining, differentiation, and antiblue staining with hematoxylin, the sections were immersed in hematoxylin for 3 min, and then differentiated with 1% hydrochloric acid for 10 s. After eosin had been impregnated for 3 min, the sections were then dehydrated by gradient. Finally, the film was sealed and photographed under a microscope. The immunohistochemical assay procedure was the same as that of our previous experiment [9 (link), 12 ]. The brain slices was incubated with the following primary antibodies: Aβ42 (1:1500, ab201060, Abcam, USA), InR (1: 500, ab5500, Abcam, USA), IRS2 (1:500, ab134101, Abcam, USA), GSK3β (1:500, ab32391, Abcam, USA), p-GSK3β (1:500, ab75814, Abcam, USA). The slices were observed under a × 20 objective lens of an optical microscope. The Aβ plaques was observed under a × 5 objective lens of an optical microscope.
5
Hippocampal Protein Signaling Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were prepared by freezing hippocampal tissues or cell homogenates with RIPA lysates containing proteases and phosphatase inhibi tors. After centrifuged at 12,000 rpm for 15 min at 4 °C, the supernatant was collected. Protein concentrations were measured by BCA kit (Bioss, China). The proteins were loaded and separated by SDS-PAGE and then transferred to PVDF membrane. The membrane was blocked by 5% defatted milk for 1 h. The membrane was then incubated with the following primary antibodies: Aβ42 (1:1500, ab201060, Abcam, USA), InR (1:1000, ab5500, Abcam, USA), p-InR (1:1000, ab60946, Abcam, USA), IRS2 (1:1000, ab134101, Abcam, USA), p-IRS2 (1:1000, ab3690, Abcam,USA),PI3K (1:1000, ab151549, Abcam, USA), Akt (1:1000, ab179463, Abcam, USA), p-Akt (1:1000, ab8805, Abcam, USA), GSK3β (1:1000, ab32391, Abcam, USA), p-GSK3β (1:1000, ab75814, Abcam, USA), GLUT1 (1:1000, ab652, Abcam, USA), GLUT3 (1:1000, ab41525, Abcam, USA),and β-actin (1:10,000, AY0573, Abways, China) at 4 °C overnight. After rinsing, the membrane was probed with secondary antibody at room temperature for 1 h. Finally, enhanced chemiluminescence reagent detection system was used to visualize the protein expression. Protein blots were quantified using ImageJ software and results were expressed quantitatively after normalizing blots with β-actin.
6
Biomarker Analysis in Metabolic Disorder
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies against 5′ AMP-activated protein kinase (AMPK) (#5831), phosphorylated AMP-activated protein kinase (#2535), Akt (#4691) P-Akt (#4060) and antirabbit IgG (#7074) were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Primary antibodies against brain-derived neurotrophic factor (BDNF) (ab108319), tropomyosin receptor kinase B (TrkB) (ab214185), insulin receptor (IR) (ab5500), glucocorticoid receptor (ab183127), and actin (ab115777) were purchased from Abcam (Cambridge, UK). The eECL western blot kit (CW0049) was obtained from Cwbio (Beijing, China). BeyoColor Prestained Color Protein Marker (P0077) was supplied by Beyotime (Nantong, China). Radio-Immunoprecipitation Assay (RIPA) buffer (PC0010), bicinchoninic acid (BCA) protein assay kit (PC0020), and other chemicals for western blotting were obtained from Solarbio (Beijing, China). The glucose assay kit was purchased from Robio Co. (Shanghai, China). Rat fast serum insulin (INS) ELISA Kit was procured from Elabscience Biotechnology Co. (Wuhan, China). STZ (552201) was obtained from Sigma (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!