The localization of PARK7, ZO-1, and the cytoskeletal actin architecture was investigated by immunofluorescence staining on frozen biopsy samples and FHs74Int cells. After repeated washing with PBS, slides were permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) for 15 minutes at RT, washed with Perm/Wash Buffer solution (BD Pharmingen), and incubated with a primary antibody specific for PARK7 (ab18257; rabbit, 1 : 1000, Abcam, Cambridge, US), ZO-1 (ab96587; rabbit, 1 : 1000, Abcam), or Alexa Fluor® 546 phalloidin (7.5 units/mL, A22283; Thermo Fisher Scientific) for 1 hour at RT. In case of PARK7 and ZO-1 staining, slides were incubated with antirabbit Alexa Fluor 568®-conjugated secondary antibody (1 : 1000, A11036; Thermo Fisher Scientific) or antirabbit Alexa Fluor 488®-conjugated secondary antibody (1 : 1000, A21206; Thermo Fisher Scientific) for 30 minutes at RT. Thereafter, the slides were washed with a Perm/Wash Buffer solution and coverslipped with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Sections were analyzed with an Olympus IX81 fluorescent microscope system.
Ab18257
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab18257 is an antibody product from Abcam. It is a polyclonal antibody raised in rabbits against a synthetic peptide within human EPAC1 protein. The antibody is suitable for use in various applications such as Western Blot and Immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm, by BD (1 mentions)
Alexa fluor 546 phalloidin, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
A11036, by Thermo Fisher Scientific (1 mentions)
A22283, by Thermo Fisher Scientific (1 mentions)
Perm wash buffer solution, by BD (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Ab96587, by Abcam (1 mentions)
Ix81 fluorescent microscope system, by Olympus (1 mentions)
Active β catenin, by Merck Group (1 mentions)
Ab8448, by Abcam (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Edta free protease inhibitor cocktail tablet, by Roche (1 mentions)
Tween 20, by Merck Group (1 mentions)
Ab90787, by Abcam (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx stain free precast gel, by Bio-Rad (1 mentions)
T per protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
6 protocols using ab18257
1
Localization of PARK7, ZO-1, and Cytoskeleton
2
Protein Analysis Using Standard Methods
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Protein analysis was performed using standard methods (Supplemental Methods).(37 ) Primary antibodies: 1:5000 total β‐catenin (detects C‐terminal region of β‐catenin; 610153; BD Biosciences, Franklin Lakes, NJ, USA), 1:1000 active β‐catenin (detects nonphosphorylated β‐catenin at Ser37 and Thr41; 05‐665; Merck Millipore, Burlington, MA, USA), 1:1000 osteopontin (ab8448; Abcam, Cambridge, UK). Park7 (ab18257; Abcam) was used as reference protein. The density of protein bands was quantified in Image J. Uncropped WB images are shown in Supplemental Fig. S2 .
3
Immunoprecipitation Protocol for Protein Complexes
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Immunoprecipitations were performed using Dynabeads (Thermo Fisher Scientific, York, UK). Antibodies (10 μg) were coupled to M270 beads according to the manufacturer's protocol. Beads were washed in C1 buffer then C1-diluted antibodies added. Buffer C2 was added and reactions incubated for 18 h at 37 °C. HB and SB washes contained 0.1% Tween-20 (Sigma-Aldrich, Dorset, UK). Before use, beads were washed in PBS containing 0.1% BSA. Lysates were prepared in kit extraction buffer supplemented with EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics Ltd., West Sussex, UK) and 100 mM NaCl. Immunoprecipitations were performed for 30 minutes at 4 °C, then beads were washed and eluted. Antibodies were ab90787, PGK1; ab18257, DJ1; and ab13579, TRF2 (Abcam, Cambridge, UK). Experiments were performed twice.
4
Kidney Protein Extraction for Western Blot
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Protein was extracted from grounded kidney tissue in T-PER protein extraction reagent (Thermo Scientific, Rockford, IL) with Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL) using a Ultra-Turrax homogenizer. Protein concentration was determined by the BCA assay (Thermo Scientific, Rockford, IL). 30μg protein were run on Mini-Protean TGX stain-free precast gel (Bio-Rad, Germany) under reducing conditions and transferred onto nitrocellulose membranes (Bio-Rad, Germany). Membranes were blocked with 5% bovine serum albumin (BSA) (Roche, Germany) in phosphate buffered saline (PBS) with 0.05% tween. Primary and secondary antibodies were diluted in PBS with 3% BSA. The antibodies used were monoclonal anti-human Napi2a (1:1000) (NBP2-42216, Novusbio), polyclonal anti-human Napi2a (1:1000) (NBP2-13328, Novusbio), polyclonal anti-human Pit2 (1:500) (HPA026540, Atlas Antibodies), polyclonal anti-mouse klotho (1:2000) (ab154163, Abcam), polyclonal anti-human Park7 (1:1000000) (ab18257, Abcam). The secondary antibodies used were anti-rabbit (1:2000) (P0448, Dako) and anti-mouse (1:1000) (P0447, Dako). Western blot quantifications were performed with ImageJ.
5
FGFR1 Western Blot Analysis
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Cells were lysed in RIPA buffer and loaded on 4%‐15% SDS gel (Bio‐Rad Laboratories, Inc). Gels were blotted on nitrocellulose membranes (Trans‐Blot Bio‐Rad Laboratories, Inc) and then incubated with anti‐FGFR1 antibody (D8E4, #9740, Cell Signaling). Signals were developed using Western Plus‐ECL (PerkinElmer). Expression of PARK7 (ab18257, Abcam) was used as a loading control.28
6
CRC Cell Apoptosis Analysis Protocol
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CRC cells were incubated with shDJ-1 for different time periods. Thereafter, they were fixed with 70% icecold ethanol at -20°C for 24 h. Cold PBS was used to wash the cells, which were then cultured with 300 uL of the staining solution (5 U/mL RNaseA and 5 ug/mL PI, BD Pharmingen, USA) for 30 min at 4°C. The results were examined with BD FASCanto II flow cytometry were from subjects with confirmed CRC who were seen at the Second Hospital Affiliated with Nanchang University from 2016 to 2018. The samples were cultured with p-MDM2 (ab170880, Abcam, 1:50), cyclin-D1 (55506T, CST, 1:500), and DJ-1 (ab18257, Abcam, 1:200) antibodies. The staining intensity of the cancer samples was scored as follows: 3 (strong staining, brown), 2 (moderate staining, yellowish-brown), 1 (weak staining, light yellow), and 0 (no staining). Intensity scores < 2 indicated low expression while intensity scores ≥ 2 indicated overexpression. Stained slides were independently examined by two researchers blinded to the clinical outcomes and patient allocation. Metastatic cancer nodules were confirmed with H&E staining.
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