Rabbit polyclonal antibodies against sortilin (Abcam, ab16640, 1:1000), rabbit monoclonal antibody against chlamydial protease/chaperone protein, CtHtrA (Huston et al., 2008 (link)) and mouse monoclonal antibodies against beta-actin (Merck Millipore, MAB1501, 1:1000) were used. Secondary antibodies were purchased from Molecular Probes (Life Technologies) and Li-Cor Bioscience. mCherry-Rab25 was obtained by performing restriction digest using restriction enzymes BamHI and EcoRI on GFP-Rab25 (Casanova et al., 1999 (link)) to obtain the open reading frame of Rab25 and subcloned into mCherry-C1 following standard protocols. Stably expressing mCherry-Rab25 HeLa cells were generated by transient transfection of mCherry-Rab25 into HeLa cells using Lipofectamine 2000 as per manufacturer's instruction (Invitrogen). Transfected cells were selected using 400 µg/ml of G418 over a period of 14 days to generate stable cell lines expressing mCherry-Rab25. pGIPZ small hairpin RNA (shRNA) plasmids (Thermo Scientific) used included non-silencing control shRNA: RHS4346; human sortilin shRNAs (shRNA #1: V2LHS_31931, shRNA #2: V2LHS_31928, shRNA #3: V3LHS_359770, shRNA #4: V3LHS_359767, shRNA #5: V3LHS_359771) were supplied by the Institute for Molecular Bioscience Life Science Automation (LISA) Facility.
Ab16640
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab16640 is a primary antibody product from Abcam. It is a rabbit polyclonal antibody targeted against a specific antigen. The antibody is supplied in a liquid format.
Automatically generated - may contain errors
Lab products found in correlation
Ab654, by Abcam (2 mentions)
Ab7291, by Abcam (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Rhs4346, by Thermo Fisher Scientific (1 mentions)
Pgipz small hairpin rna shrna plasmids, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Mab1501, by Merck Group (1 mentions)
Ab32575, by Abcam (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Ab77528, by Abcam (1 mentions)
Ab30871, by Abcam (1 mentions)
Ab13492, by Abcam (1 mentions)
Ab92726, by Abcam (1 mentions)
Af1404, by R&D Systems (1 mentions)
Sc 548, by Santa Cruz Biotechnology (1 mentions)
Sc 20981, by Santa Cruz Biotechnology (1 mentions)
β actin a5316, by Merck Group (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab45174, by Abcam (1 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
13 protocols using ab16640
1
Generating mCherry-Rab25 HeLa Cell Line
2
Immunofluorescent Staining of Frozen Aortic Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fresh aortic tissue was snap-frozen, and the OCT embedded gel was completely covered on it, then serial frozen sections were performed. Frozen tissue sections were dried at room temperature for 15 minutes. The sections were then incubated with 1% bovine serum albumin (BSA) for 1 hour at 37°C. The primary antibody was added, which was diluted as required and incubated for 16 hours. After washing the sample in PBS, a secondary fluorescent antibody was added and incubated for 1 hour in the dark. The image was taken under an inverted microscope (Olympus). The antibodies used are: Rabbit polyclonal to Sortilin 1:100 (Abcam Cat# ab16640, RRID: AB_2192606); Rabbit monoclonal to alpha smooth muscle Actin 1:100 (Abcam Cat# ab32575, RRID: AB_722538).
3
Exosome Molecular Profiling by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and exosomes were lysed in lysis buffer [50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100μg/ml phenylmethylsulphonyl fluoride, 0.5 μg/ml leupeptin, and 1 μg/ml aprotinin]. Protein concentrations were determined using a Micro BCA™ protein assay kit (Pierce Biotechnology). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBS containing 0.1% Tween-20 for 2 h at room temperature and incubated with the appropriate primary and secondary antibodies. Antibodies against CD63 (HPA010088, 1/1000) and β-actin (A 5316, 1/10000) were purchased from Sigma-Aldrich. Antibodies against YKL40 (ab77528, 1/1000), sortilin (ab16640, 1/1000), CD9 (ab92726, 1/1000) and HSP90 (ab13492, 1/1000), TSG101 (ab30871, 1/1000) were obtained from Abcam. Antibodies against TrkA (AF175, 1/200) TrkB (MAB397, 1/200), TrkC(AF1404, 1/200) and P75NTR (AF1157, 1/1000) were purchased from R&D System. Antibodies against NT3 (5237SC, 1/1000), Sox2 (3579S, 1/1000), Oct4 (2750S, 1/1000) and flotillin (18634S, 1/1000) were purchased from Cell Signaling Technology. Antibodies against NGF (sc-548, 1/200) and BDNF (sc-20981, 1/200) were purchased from Santa Cruz Biotechnology.
4
Hepatocyte Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hepatocytes were harvested and washed twice in ice-cold PBS. Total cellular protein was extracted using a protein extraction kit according to the manufacturer's instructions. Aliquots of 60 μg total protein were denatured for 5 min at 100°C and loaded on 10% sodium dodecyl sulfate polyacrylamide gels and electro-transferred onto polyvinylidene difluoride (PVDF ) membranes. After incubation with blocking buffer, membranes were exposed to polyclonal GRP78 (78 kD, 1:250, sc-376768; Santa Cruz, CA), SREBP-1c (68 kD, 1:1000; NB100-2215; Novus, USA), FAS (273 kD, 1:1000; C2065; Cell Signaling, Danvers, MA, USA), SORT1 (85 kD,1:1000; ab16640, Abcam, Cambridge, MA), P65 (65 kD, 1:1000; D14E12; Cell Signaling; Danvers, MA, USA), ACC1 (265 kD, 1:1000; ab45174, Abcam, Cambridge, MA), CHOP (27 kD, 1:1000; L63F7; Cell Signaling, Danvers, MA, USA), PCSK9 (65–80 kD, 1:1000; 85813, Cell Signaling; Danvers, MA, USA), SCD1 (37 kD, Cell Signaling; Danvers, MA, USA). After washing and incubation with secondary antibody, immunoreactive bands were detected by enhanced chemiluminescence (Beyotime, China) solution. The imprinting was exposed with X-ray film to radiograph the spectral band, and the gray value of the spectral band was measured with bandscan software version 5.0 (prozyme Inc, San Leandro, CA). Protein abundance is reported relative to that of β-actin as a ratio of optical densities.
5
Antibody Panel for Endosomal Trafficking
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in this study are as follows: SNX1 [clone 51; 611482; BD Bioscience; immunofluorescence (IF) 1:200], GLUT1 (ab40084; Abcam; IF 1:200), Golgin97 (clone CDF4; A-21270; Thermo Fisher Scientific; IF 1:400), VPS26 (ab23892; Abcam; IF 1:200), VPS35 (ab10099; Abcam; IF 1:200), VPS35 [ab97545; Abcam; IF 1:200), VPS35 [ab157220; Abcam; western blotting (WB) 1:1000], VPS29 (ab98929; Abcam; WB 1:200), FAM21 (gift from Daniel D. Billadeau, Mayo Clinic, Rochester, MN; IF 1:400), EEA1 (N-19; sc-6415; Santa Cruz Biotechnology; IF 1:200), TGN46 (AHP500G; Bio-Rad Laboratories; IF 1:200), anti-Myc (gift from Harry Mellor, The University of Bristol, UK; IF 1:500), LAMP1 (DSHB Hybridoma Product; H4A3; deposited to the DSHB by August, J.T./Hildreth, J.E.K.; IF 1:500), LAMP1 (ab24170; Abcam; IF 1:200), mouse EEA1 (610457; BD Bioscience; IF 1:200), CI-MPR (ab124767; Abcam; WB 1:1000, IF 1:200), Itgβ1 (TS2/16; IF 1:200), SNX6 (Clone D-5; 365965; Santa Cruz Biotechnology; WB 1:500), PMP70 (PA1-650; Invitrogen; IF 1:200), WASH1 (gift from Daniel D. Billadeau; IF 1:400), C16orf62 (PA5-28553; Pierce; IF 1:200), sortilin (ab16640; Abcam; WB 1:1000), cathepsin D (21327-1-AP, Proteintech; WB 1:1000), SNX5 (ab180520; Abcam; WB 1:500) and β-actin (A1978; Sigma-Aldrich; WB 1:2000).
6
Western Blot Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from tissues or cell lines were extracted using the RIPA lysis buffer (Beyotime, Shanghai, China). 20 μg proteins were separated in 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche, Mannheim, Germany), then the transfected membranes were blocked in Tris-buffered saline containing 5% skimmed milk powder for 2 h. Next, the membranes were incubated with Anti-SORT1 (1 μg/ml, ab16640, abcam), Anti-Beclin-1 (1:1000, ab210498, abcam), Anti-LC3B (1:2000, ab192890, abcam) or Anti-p62 (1:1000, ab56416, abcam) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein signals was visualized by ECL chemiluminescent reagent (Millipore, MA, USA).
7
Protein Expression Profiling of Adipose and Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates of adipose and brain tissues were prepared using radioimmunoprecipitation assay lysis buffer (Beyotime, Jiangsu, China) containing protease inhibitors (Sigma-Aldrich, St. Louis, Missouri, USA). Ten microliters of the homogenates were kept for total protein level analysis using bicinchoninic acid (BCA) kits (Beyotime) according to the manufacturer’s instructions. Tissue lysate, isolated cell membrane proteins and Co-IP samples were analyzed by 10% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred to polyvinylidene fluoride membrane. The membrane was first blocked in Tris-buffered saline containing 0.1% Tween-20 and incubated with appropriate primary (goat anti-HAP1 antibody (sc-12556, 1:1000; Santa Cruz), goat anti-GLUT4 antibody (sc-1608, 1:1000; Santa Cruz), rabbit anti-GLUT4 antibody (ab654, 1:1000; Abcam), rabbit anti-sortilin antibody (ab16640, 1:1000; Abcam), rabbit anti-Na/K-ATPase antibody (3010, 1:1000; CST)) and secondary antibodies (rabbit anti-goat IgG-HRP antibody (sc-2768, 1:2000; Santa Cruz), goat anti-rabbit IgG-HRP antibody (sc-2004, 1:2000; Santa Cruz)). The membrane was developed with Immobilon Western (P90719; Millipore, Burlington, Massachusetts, USA) and examined via Bio-Rad ChemiDoc MP system (Bio-Rad, Hercules, California, USA). Captured images were quantified using ImageJ software (http://rsb.info.nih.gov/ij/ ).
8
Antibody Procurement for Protein Analysis
9
Co-Immunoprecipitation of Adipose Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Co-IP was performed as described previously.30 (link) Briefly, cell lysates were prepared from white adipose tissue from mouse epididymal adipose pad or HEK293 cells. The lysates were pre-cleared by adding protein A/G magnetic beads (Bimake, Houston, Texas, USA) in lysis buffer, the mixture was rotated for 30 min in 4°C and then magnetic beads separated; the supernatant were kept for Co-IP experiment. The pre-cleared lysates (500 µg) were incubated with 1 µg of goat anti-HAP1 antibody (sc-12556; Santa Cruz, Dallas, Texas, USA) or goat anti-GLUT4 antibody (sc-1608, Santa Cruz) or rabbit anti-GLUT4 antibody (ab654; Abcam, Cambridge, UK) or rabbit anti-sortilin antibody (ab16640, Abcam) at 4°C for 12 hours, and then incubated with protein A/G beads for 1 hour. The beads were washed five times with the lysis buffer and boiled in loading buffer for western blot. For negative control, goat and rabbit IgG and untransfected HEK293 were used for validation.
10
Antibody Validation for AKT/GLUT4 Analysis
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!