Propidium iodide and TO-PRO®-3 iodide were purchased from Sigma-Aldrich® (St. Louis, MO) and Life Technologies™ Europe B.V. (Bleiswijk, Netherlands), respectively. Fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal secondary antibody anti-human IgG1-Fc (Ab50473), rabbit polyclonal anti-HER 2 (ab2428), anti-pHER-2 (ab47263), anti-ACTIN (ab1801) antibodies and horseradish peroxidase (HRP)-conjugated goat polyclonal secondary anti-Rabbit IgG Fc (ab97200) were obtained from Abcam® (Cambridge, UK). FITC-conjugated human IgG1 isotype control (ANC-295-040) and human IgG1 isotype control (ANC-295-010) were purchased from Adipogen® International (Liestal, Switzerland).
Ab1801
Manufactured by Abcam
Sourced in United Kingdom, Japan, United States
Ab1801 is a primary antibody designed for use in immunohistochemistry applications. It targets a specific protein of interest. The core function of this product is to facilitate the detection and visualization of the target protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software v 4, by Bio-Rad (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Ab6721, by Abcam (4 mentions)
Complete mini, by Roche (4 mentions)
Block ace reagent, by Sumitomo Pharma (3 mentions)
Ab16066, by Abcam (3 mentions)
Ab7260, by Abcam (3 mentions)
Supersignal west pico ecl, by Thermo Fisher Scientific (3 mentions)
Exoab cd63a 1, by System Biosciences (3 mentions)
Stain free tgx gradient gels, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Abcam (2 mentions)
Enhanced chemi luminescence (ecl), by Avantor (2 mentions)
C digit blot scanner, by LI COR (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ab9485, by Abcam (2 mentions)
Irdye 800cw, by LI COR (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Goat anti mouse igg h l hrp conjugated secondary antibodies, by Bio-Rad (2 mentions)
Ab175472, by Abcam (2 mentions)
Ab5076, by Abcam (2 mentions)
41 protocols using ab1801
1
Antibody Detection and Quantification
2
Western Blot Analysis of PIWIL Proteins
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Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris- HCl, pH 8.0) supplemented with protease Inhibitor Complete Mini (Roche, 000000011836170001) on ice for 15 min. The cell lysates were rotated at 4 °C for 30 min and the insoluble material was removed by centrifugation at 15,000 g for 15 min. Protein concentrations were determined by Bio-Rad Protein Assay using bovine serum albumin (BSA) as the standard. Following the normalization of protein concentrations, lysates were mixed with an equal volume of 2X Laemmli sample buffer and incubated for 5 min at 95 °C prior to separation by SDS PAGE on stain-free TGX gradient gels (Bio-Rad). Following SDS-PAGE, the proteins were transferred to polyvinylidene fluoride membranes (300 mA for 90 min at 4 °C). The membranes were then blocked with BSA (Sigma-Aldrich) dissolved in PBS/Tween-20 (3% BSA, 0.5% Tween-20 for 1–2 hours), followed by immunoblotting with the primary antibody specified for each experiment for PIWIL2 (Abcam, ab181340 diluted at 1:1000), PIWIL4 (Abcam ab 111714, diluted at 1:1000), and beta Actin (Abcam ab1801, diluted at 1:1000). After the washing steps, the membranes were incubated with goat anti-rabbit IgG (H + L) HRP-conjugated secondary antibodies (Bio-Rad) and detected using ECL (Amresco). Densitometry was performed using Image Lab software v. 4.1 (Bio-Rad).
3
Extracellular Vesicle Protein Profiling
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EV marker and immuno-related proteins in lysates from EVs and EECs were detected through the use of SDS-PAGE. Separated proteins transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) were blocked with Block Ace reagent (DS Pharma Biomedical, Osaka, Japan), and the membranes were incubated with the following antibodies; rabbit polyclonal anti-human CD63 antibody (1:2000, EXOAB-CD63A-1, System Biosciences), rabbit polyclonal anti-human HSP70 antibody (1:2000, EXOAB- HSP70A-1, System Biosciences), rabbit polyclonal anti-bovine CTSC (1:2000, ab182904, abcam, Tokyo, Japan), rabbit polyclonal anti-pocine IL6 (1:2000, ab193853, abcam), or rabbit polyclonal anti-human ACTB (1:5000, ab1801, abcam). The membranes were then incubated with secondary antibody, horseradish peroxidase labeled goat anti-rabbit IgG (1:5000, Vector Laboratories, Burlingame, CA, USA), and immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, USA). Signals were detected using C-DiGit Blot Scanner (LI-COR) and then band density was assessed with Image Studio DiGit software (version 5.2)37 (link).
4
Decidual and Placental C3 Expression
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To determine expression of C3, C3b, and iC3b, decidual or placental tissues (n = 3 each day) were prepared in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1 mM Na3VO4, and 50 mM NaF). Decidual and placental tissue lysates (10 μg/lane) were separated through 12.5% SDS-PAGE and were then transferred onto a total of three polyvinylidene difluoride (PVDF) membranes (Millipore, Milford, MA). After blocking with Block Ace reagent (DS Pharma Biomedical, Osaka, Japan), membranes were incubated with rabbit monoclonal anti-human C3 antibody (1:2000, ab200999, Abcam, Tokyo, Japan), rabbit polyclonal anti-mouse CD11b antibody (1:500 dilution, 0.5 mg/ml, ab75476, Abcam), or rabbit monoclonal anti-human ACTB antibody (for internal control, 1:1000, ab1801, Abcam). Immunoreactive bands were detected using enhanced chemiluminescence (Millipore) after incubation with horseradish peroxidase-labeled SAP solution (APRO life Science, Inc., Tokushima, Japan).
5
Quantifying HK2 Protein Expression
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Cell protein was isolated using RIPA buffer (Beyotime, Shanghai, China) and quantified using BCA assay kit (Sigma). Protein samples (20 μg) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Solarbio, Beijing, China). Next, the membranes were blocked in 5% skim milk, and then interacted with primary and secondary antibodies, followed via interacting with BeyoECL Plus kit (Beyotime). The antibodies were shown as: anti-HK2 (ab209847, 1:500 dilution, Abcam), and anti-β-actin (ab1801, 1:1000 dilution, Abcam), as well as horseradish peroxidase-conjugated IgG (ab205718, 1:20,000 dilution, Abcam). β-actin was used as a loading reference. HK2 protein level was normalized to the control group.
6
Quantifying Brain-Derived Neurotrophic Factor
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To quantify BDNF levels, protein extracts (10 μg) were separated on 4–12% Nupage BisTris gels (Invitrogen, Carlsbad, CA). The proteins were transferred onto a Polyvinylidene fluoride membrane using a semidry transblotter (Bio-Rad). Blots were then probed with a BDNF antibody (1:1000, Cat. No. sc-546, Santa Cruz Biotechnology Inc., Dallas, Texas, USA). Secondary goat anti-rabbit 800CW antibody was used for protein detection (Licor Odyssey System, LiCor, Lincoln, NE). Quantifications were normalized to actin (1:1000, Cat. No. ab1801, Abcam, Cambridge, UK) and valosin-containing protein (1:5000, #ab11433, Abcam).
7
Detecting DNA Damage and Repair
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Mitomycin C (MMC, M0503), diepoxybutane (DEB, ref. 202533-16) 5-bromo-2-deoxyuridine (BrdU, B5002) and Hoechst 33258 (B2883) and IGEPAL (I8896) were from Sigma (Madrid, Spain). Ethidium monoazide bromide (EMA, e1374) and Sytox Green (S7020) were from Thermofischer Scientific (Barcelona, Spain). PARP inhibitor Olaparib (S1060) was from Selleckchem (Munich, Germany). Propidium iodide (P3566) and RNAse (12091021) were from Invitrogen (Barcelona, Spain). Colcemid was from Gibco (Barcelona, Spain). Antibodies used included: mouse monoclonal to CDK5RAP3 (ab57817), rabbit polyclonal to BRCA2 (ab123491), mouse monoclonal to RPA32 (ab2175), rabbit polyclonal to actin (ab1801), rabbit polyclonal to GAPDH (ab9485) and mouse monoclonal to vinculin (ab18058) were from Abcam (Cambridge, UK). Rabbit polyclonal to RAD51 (SC8349) was from Santa Cruz biotechnology (Heidelberg, Germany).
8
Immunoblotting Analysis of Brain PrP
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Brain homogenates analyzed without
proteinase K (PK) treatment were diluted from 20% to 2.5% and boiled
in Tris-buffered saline, pH 8, and sample buffer (Life Technologies)
with 0.5% β-mercaptoethanol. Brain homogenates treated with
PK were diluted to 6% in the presence of 0.1 M Tris, pH 8.3, 1% Triton
X-100, 1% sodium deoxycholate, then digested with 125 μg/mL
PK for 1 h at 37 °C and then boiled in an equal volume of sample
buffer. Portions of each sample (4 μL) were separated by SDS-PAGE
on a 10% Bis–Tris gel with MES running buffer (Life Technologies)
and then transferred to an Immobilon PVDF-FL membrane (Millipore)
by wet transfer using Towbin’s buffer. Samples were probed
for PrP with mouse monoclonal antibody 6D11 (Covance Inc.) at a dilution
of 1:15 000 in TBST buffer for 1 h. 6D11 recognizes residues
93–109 of mouse PrP. Antivimentin antibody (Origene #TA307358)
was used at 1:2000. Anti-apoE antibody (Calbiochem #178479) was used
at 1:1000 and antiactin (abcam #ab1801) was used at 1:1000. Secondary
antibodies (Li-Cor Biosciences) were IRDye 800CW or IRDye 680CW anti-mouse,
anti-rabbit or anti-goat, used at a 1:15 000 dilution in TBST
buffer for 40 min. Imaging was performed with an Odyssey imaging system
(Li-Cor) using the 800 and 680 channels.
proteinase K (PK) treatment were diluted from 20% to 2.5% and boiled
in Tris-buffered saline, pH 8, and sample buffer (Life Technologies)
with 0.5% β-mercaptoethanol. Brain homogenates treated with
PK were diluted to 6% in the presence of 0.1 M Tris, pH 8.3, 1% Triton
X-100, 1% sodium deoxycholate, then digested with 125 μg/mL
PK for 1 h at 37 °C and then boiled in an equal volume of sample
buffer. Portions of each sample (4 μL) were separated by SDS-PAGE
on a 10% Bis–Tris gel with MES running buffer (Life Technologies)
and then transferred to an Immobilon PVDF-FL membrane (Millipore)
by wet transfer using Towbin’s buffer. Samples were probed
for PrP with mouse monoclonal antibody 6D11 (Covance Inc.) at a dilution
of 1:15 000 in TBST buffer for 1 h. 6D11 recognizes residues
93–109 of mouse PrP. Antivimentin antibody (Origene #TA307358)
was used at 1:2000. Anti-apoE antibody (Calbiochem #178479) was used
at 1:1000 and antiactin (abcam #ab1801) was used at 1:1000. Secondary
antibodies (Li-Cor Biosciences) were IRDye 800CW or IRDye 680CW anti-mouse,
anti-rabbit or anti-goat, used at a 1:15 000 dilution in TBST
buffer for 40 min. Imaging was performed with an Odyssey imaging system
(Li-Cor) using the 800 and 680 channels.
9
VSMC Protein Expression Analysis
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After cell treatment, VSMCs were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer (Biotech, Shanghai, China). After one freeze/thaw cycle, lysates were centrifuged. Protein concentration was determined by a BCA protein assay (Biotech, Shanghai, China) using bovine serum albumin as the standard. A quantity amounting to 10 μg of protein sample was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to an ECL nitrocellulose membrane (Millipore). Incubating the membrane in Superblock (Pierce) for 1 h blocked nonspecific binding. Membranes were then incubated overnight at 4°C in primary antibodies, PPARγ1, Cyclin D1, Cyclin B1/cdc2 and β-actin (AbCam: ab8924, ab95281, ab7959, ab1801). All primary antibodies dilution was 1:1000 in each reaction. The blots were washed three times with TBST buffer and then incubated for 1 h at room temperature with anti-rabbit secondary antibody conjugated with horseradish peroxidase. Western blot analysis was conducted according to standard procedures using Supersignal chemiluminescence detection substrate (Pierce).
10
Western Blot Analysis of PPARγ and Klotho
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Cells were lysed in radioimmunoprecipitation buffer supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Inc.). The protein concentration was determined using the BCA protein assay. Proteins were subjected to SDS-polyacrylamide gel electrophoresis and subsequently transferred onto a nitrocellulose membrane. Following blocking with 3% bovine serum albumin, the membrane was incubated with primary antibodies, including anti-PPARγ (1:500; ab66343; Abcam, Cambridge, UK), anti-Klotho (1:1,000; ab203576; Abcam) and anti-β-actin (1:500; ab1801; Abcam) overnight at 4°C. Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000; BM2006; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 1 h at room temperature. Protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Thermo Fisher Scientific, Inc.), followed by exposure of the membranes to X-ray film. Image J software (version 1.140; US National Institutes of Health, Bethesda, MD, USA) was used to quantify the blot intensity.
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