GLP-1R internalization and recycling in CHO-SNAP-GLP-1R cells was measured by surface labeling with anti-GLP-1R antibody or the SNAP-tag probe Lumi4-Tb (Cisbio). For antibody labeling, after treatments at 37 °C, cells were placed on ice to arrest further endocytosis before fixation, and the surface GLP-1R was detected by ELISA with monoclonal anti-human GLP-1R antibody68 (link) (Mab 3F52, Developmental Studies Hybridoma Bank (DSHB), 1/100) plus horseradish peroxidase (HRP)-conjugated rabbit anti-mouse secondary (ab97046, Abcam, 1/5,000). 3,3′,5,5′- tetramethylbenzidine (TMB) substrate was added and the absorbance was read at 450 nm after 1 M HCl addition. For Lumi4-Tb, cells were labeled at 40 nM at 4 °C, followed by time-resolved (TR) fluorescence measurement in a Molecular Devices i3x (excitation 335 nm, emission 620 nm). Residual surface expression was determined from peptide- vs. control-treated wells. Recycling was measured by comparing the surface receptor immediately after internalization vs. a further period of recycling with agonist washed off and replaced with 10 μM exendin(9-39); and recycling calculated as percentage recovery of surface GLP-1R in the recycling plate vs. surface receptor loss in the internalization plate.
Ab97046
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab97046 is a laboratory equipment product manufactured by Abcam. It is a device used for scientific research and analysis purposes. The core function of this product is to [product description]. Further details on the intended use or specifications of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab97051, by Abcam (11 mentions)
α tubulin, by Merck Group (4 mentions)
Ecl detection system, by Cytiva (4 mentions)
Ab6721, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ab205718, by Abcam (3 mentions)
Anti mybbp1a, by Proteintech (2 mentions)
Chemidoc xrst, by Bio-Rad (2 mentions)
Ab6308, by Abcam (2 mentions)
Pierce 1step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (2 mentions)
Ab97100, by Abcam (2 mentions)
T9026, by Merck Group (2 mentions)
Ab32360, by Abcam (2 mentions)
Ab9572, by Abcam (2 mentions)
Ab34843, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Ab9485, by Abcam (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Ab52866, by Abcam (2 mentions)
47 protocols using ab97046
1
Measuring GLP-1R Internalization and Recycling
2
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots were performed as previously described elsewhere. Membranes were incubated with the following primary antibodies: anti‐MYBBP1A (Proteintech #14524‐AP, Rosemont, IL, USA ), anti‐PGC1α (Abcam #ab54481, Cambridge, UK), anti‐SGLT1 (Abcam #ab14685), anti‐p38 MAPK (Cell Signaling #9212, Danvers, MA, USA), and anti‐phospho‐p38 MAPK (T180/Y182) (Cell Signaling #9215). α‐Tubulin (Sigma #T9026) was used as a loading control. Horseradish peroxidase‐labeled rabbit anti‐mouse (Abcam #ab 97046) and goat anti‐rabbit (Abcam #ab 97051) secondary antibodies were used. The proteins were detected using an ECL detection system (Amersham Biosciences) and Bio‐Rad ChemiDoc XRST (Berkeley, CA, USA).
3
Protein Extraction and Immunoblotting for USP21 in Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich; EMD Millipore) as previously described (22 (link)–24 (link)). Cells were centrifuged at 4°C for 10 min at 16,000 × g. Protein concentrations were determined by the Bradford assay (25 (link)). Aliquots containing 20 µg of total protein were separated by 10% sodium dodecyl-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (1:5,000, mouse monoclonal; A5316; Sigma-Aldrich; EMD Millipore). Appropriate secondary antibodies (1:3,000; rabbit anti-goat; ab6741; 1:5,000; rabbit anti-mouse; ab97046; Abcam, Cambridge, MA, USA) conjugated to horseradish peroxidase and enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK) were used to detect the bound primary antibodies. Co-IP was performed with cell lysate (500 µg) incubated with USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc.), relA (1:1,000; rabbit polyclonal; sc-372; Santa Cruz Biotechnology, Inc.) or non-specific-IgG antibodies (1:1,000; rabbit IgG, monoclonal; ab172730; Abcam) using µMACS™ Protein A/G MicroBeads and MACS® Separation Columns according to the manufacturer's protocol (Miltenyi Biotec, Auburn, USA).
4
Collagen Production in Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The production of collagen type I by 46BR.1N fibroblasts was evaluated by ELISA as previously reported [30 (link)]. Briefly, cells were settled in 96-well plates and incubated with 5, 10, or 20 µg/mL PEE for 48 h. Thereafter, cells were fixed with 3.7% paraformaldehyde, blocked with BSA, probed with mouse anti-human collagen type I (ab6308, Abcam, Cambridge, UK), and then with HRP-conjugated rabbit anti-mouse IgG (ab97046, Abcam), incubated with Pierce 1Step™ Ultra TMB ELISA Substrate Solution (Thermo Fisher Scientific), blocked with 2 M sulfuric acid, and read at 620 nm in the microplate reader.
5
IGFBP-2 and IGFBP-5 Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Conditioned media (CM: 1 ml) were freeze dried and stored at −20°C prior to analysis. Anti-hIGFBP-2 (MAB6741) and -5 (MAB8751) were from R & D Systems. MAB 6741 has < 1% cross reactivity with IGFBP-5; MAB 8751 has no cross reactivity with IGFBP-2. Anti-β-actin was from Santa Cruz Biotechnology. HRP conjugated rabbit anti-mouse was from Abcam (ab97046). Western blot protocols have been reported previously [54 (link)]. Ligand blotting with monobiotinylated IGF-2 (AMU010- GroPep) and streptavidin-HRP conjugate was as originally described [55 (link)]. Western and ligand blots were developed with Super-Signal® West Femto Maximum Sensitivity Substrate (PN34095; Fisher Scientific) and images processed using Gel-Doc imager (Bio-Rad).
6
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction and western blotting was performed using standard procedures. In brief, 30 μg protein per sample was separated by Bolt® Bis‐Tris 4–12% SDS/PAGE (Thermo Fisher) and blotted onto polyvinylidene fluoride (PVDF) by iBlot® (Invitrogen, Carlsbad, CA, USA) and blocked by 4% skim milk in 2% Tris‐buffered saline/Tween. Primary antibodies used in this study were as follows: 2A peptide 1 : 2000 ABS31 (Millipore, Billerica, MA, USA), SV40LT 1 : 400 Pab416 (Abcam, Cambridge, UK), and β‐actin 1 : 10 000 AC‐15 (Abcam). Secondary horseradish peroxidase (HRP) antibodies used in this study were as follows: goat anti‐rabbit (AP156P; Millipore) and rabbit anti‐mouse (ab97046; Abcam).
7
Immunoblotting Analysis of STAT1 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell pellet was lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) with 1X proteCEASE-50 (#427P;G-Biosciences, St.Louis, MO,USA) 1μM PMSF. Protein was quantified by Bicinchoninic acid (BCA) assay. The protein was resolved on SDS-PAGE and transferred on PVDF membrane. Membrane was kept for blocking in 5% skimmed milk in 1X TBS-Tween20. Thereafter, the membrane was incubated in primary antibody (1:1000) overnight. HRP-conjugated secondary antibody incubation was used for 1 hour and then membrane was washed thrice with 1X TBST and developed by using Super-Signal developing reagent as HRP substrate. The anti-STAT1 (#9172 Cell Signaling technology), anti-phospho-Y701-STAT1 (#9167 Cell Signaling technology), anti-SOCS-5 (#sc-5607, Santa cruz biotechnology) anti-JEV NS1 (#ab41651; Abcam) and anti-β-tubulin (#ab6046; Abcam) primary antibodies were used after diluting in 5% BSA in TBST buffer. HRP-conjugated anti-rabbit (#ab6721-1; Abcam) and anti-mouse (#ab97046, Abcam) secondary antibody was used in 1:50,000 dilutions.
8
SARS-CoV-2 Spike Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sucrose cushion-purified viruses were diluted with PBS, and the protein amounts were then measured by BCA assay according to the manufacturer’s protocol (Pierce™ Micro BCA™ Protein-Assay, Thermo Fisher, Waltham, MA, USA). Samples were treated with 2-mercaptoethanol and sample reducing buffer and then incubated at 95 °C for 5 min. The proteins were separated by SDS–PAGE, transferred to an Immobilon-P PVDF membrane (Millipore Sigma, Munich, Germany) and blocked with 5% milk in TBS-T. Primary antibodies were allowed to bind overnight at 4 °C, after which the membranes were washed in TBS-T and incubated with secondary antibodies for an hour at RT. Upon another wash, images were acquired by a Chemostar PC ECL & Fluorescence Imager (Intas Science, Goettingen, Germany). Anti-SARS-CoV-2 spike (1A9, GeneTex, Irvine, CA, USA), anti-HA (kindly provided by W. Gerhard from Philadelphia), anti-MCK-2 (kindly provided by Stipan Jonjic) and IE1 (IE 1.01, CapRi, Rijeka, Croatia) were used as primary antibodies. Anti-rabbit IgG (ab205718, Abcam, UK) and anti-mouse IgG (ab97046, Abcam, UK) were used as secondary antibodies.
9
Salivary Histatin-1 Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Salivary protein concentration was measured by bicinchoninic acid (BCA) kit (Solarbio, China). Salivary total protein (20 μg) underwent electrophoresis in 18 % gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) using Mini-PROTEAN Tetra Cell System (Bio-Rad, USA). Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes. The total protein load was checked visually by Ponceau staining before immuno-detection. The membranes were blocked overnight at 4 °C with 10 % skimmed milk in Tris-buffered saline (TBS, pH 7.4), followed by washing with TBS buffer containing 0.05 % tween20 (TBST). Membranes were incubated with the primary antibody specific for histatin-1 protein (ab70024, Abcam, UK) and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) monoclonal antibody (TA-08, ZSGB-Bio, China) at room temperature for 1 h. And the secondary antibodies used were rabbit polyclonal anti mouse immuno-globin G-horseradish peroxidase (IgG-HRP) (ab97046, Abcam) with the same condition, then the excess ones were removed. After that, the immuno-reactive bands were visualized by Odyssey scanner (Gene Company Limited, Hong Kong), and western blotting bands were quantified using Image J software (http://www.rsbweb.nih.gov/ij/ ).
10
Western Blot Analysis of PPARγ and GFP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentrations were determined using the Bradford assay (Thermo Fisher Scientific). Samples were subjected to SDS-PAGE in polyacrylamide gels (NuPAGE 4–12% Bis–Tris Protein Gels, Thermo Fisher Scientific) and transferred onto PVDF membranes (Thermo Fisher Scientific, #PI-88518) using NuPAGE Novex system (Thermo Fisher Scientific). Proteins were detected using primary antibodies against PPARγ (Santa Cruz Biotech, sc-7273, 1:1,000) and GFP (Abcam, ab111258, discontinued, 1:2,000), secondary anti-goat (Thermo Fisher Scientific, #31402, 1:15,000) and anti-mouse peroxidase-conjugated antibodies (Abcam, ab97046, 1:15,000), and the SuperSignal Femto ECL kit (Thermo Scientific) according to the manufacturer’s protocol. Equal protein loading was verified using HRP-conjugated α-β-actin antibody (Santa Cruz, sc-47778).
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!