For the in vitro experiments, LPS from E. coli LPS (E. coli, Sigma Aldrich, St Louis, MO, USA) was used. Most of the antibodies used for Western blots were from Abcam (Abcam, Cambridge, UK) anti Prx pathway (Trx1, TrxR, and Prx1) cocktail (ab184868), anti peroxiredoxin 2 (ab109367), anti peroxiredoxin 3 (ab73349). Antibodies anti β-actin were from Sigma (A1978) (Sigma Aldrich, St Louis, MO, USA).
Ab109367
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab109367 is an antibody that can be used for the detection of a specific target protein in various experimental applications. The product's core function is to provide a tool for researchers to identify and study the target protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (2 mentions)
Ma1 048, by Abcam (2 mentions)
Starbright blue 700, by Bio-Rad (2 mentions)
Hm 2016, by Hycult Biotech (2 mentions)
Anti β actin antibody, by Bio-Rad (2 mentions)
Irdye 800cw, by LI COR (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab32572, by Abcam (2 mentions)
Ab46545, by Abcam (2 mentions)
Ab41906, by Abcam (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Ab73349, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Ab42824, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
Ab40766, by Abcam (1 mentions)
Ab252421, by Abcam (1 mentions)
Ab76523, by Abcam (1 mentions)
15 protocols using ab109367
1
LPS-induced Peroxiredoxin Pathway
2
Western Blot Analysis of RSV Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as previously described (15 (link)). Briefly, the protein concentrations of the RSV infection serum samples (15 patients with acute- and convalescence-phase RSV infection, 15 control) and mouse lung homogenates (10 mice with acute- and convalescence-phase RSV infection, 10 control) were determined using a BCA kit (Beyotime Biotechnology, China). Protein samples (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at constant voltage in Tris-glycine buffer, and then transferred onto polyvinylidene difluoride membranes at constant current. The membranes were washed thrice in Tris-buffered saline containing 0.1% TWEEN 20 (TBST), blocked with 10% bovine serum albumin for 1 h at room temperature, and incubated with primary antibodies against triosephosphate isomerase 1 (TPI1; ab28760, Abcam, USA), peroxiredoxin 2 (PRDX2; ab109367, Abcam), bisphosphoglycerate mutase (BPGM; ab97497, Abcam), and cofilin 1 (CFL1; ab42824, Abcam) at 4°C overnight. The next day, membranes were washed three times in TBST and incubated with secondary antibody for 1 h at room temperature. After three washes in TBST, protein signals were visualized with ECL (Beyotime Biotechnology, China) and exposed to X-ray film. The band intensities were quantitated using ImageJ software (Version 2.0; NIH, USA) and normalized to GAPDH.
3
Western Blot Characterization of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three samples from each of the four sample groups were randomly selected for verification by Western blot. Protein was fractionated by SDS-PAGE (120 V loading, 160 V for ~80 min). Proteins were transferred to a nitrocellulose membrane using a wet transfer at 100 V for 70 min. After incubation with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST; 50 mM Tris, 150 mM NaCl, pH 7.4, 0.1% Tween-20) for 30–60 min, the membrane was washed 4 × 4 min with TBST and incubated overnight with antibodies against calcium/calmodulin dependent protein kinase IIα (CAMKIIα; Thermo MA1-048; 1:5000), peroxiredoxin-2 (PRDX2; abcam ab109367; 1:10,000), or fatty acid-binding protein, heart isoform (H-FABP; Hycult Biotech HM 2016; 1:1000) at 4 °C. Membrane was washed 4 × 4 min with TBST and incubated with a 1:10,000 dilution of fluorescent-labeled anti-mouse (StarBright Blue 700; Bio-Rad Laboratories) or anti-rabbit (IRDye 800CW; Li-Cor Biosciences) secondary antibodies for 30–60 min. For β-actin blots, membrane was incubated with a rhodamine-conjugated anti-β-actin antibody (Bio-Rad 12,004,164; 1:10,000) overnight (no secondary antibody necessary). Blots were washed 4 × 4 min with TBST prior to imaging using a ChemiDoc MP imaging system (Bio-Rad). ImageLab software (Bio-Rad, version 6.0) was used for band quantification.
4
Bile Protein Analysis in Choledocholithiasis and HCC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 20 μg of proteins from each patient’s bile (choledocholithiasis, n = 14; HCC, n = 12) were separated by SDS-gel electrophoresis and transferred to PVDF membranes (Millipore, USA). The membranes were then incubated overnight at 4°C with primary antibodies, including anti-PRDX2 (1:1000, Abcam, ab109367), anti-CyclinD1 (1:1000, Abcam, ab16663), anti-cMYC (1:1000, Abcam, ab32072), anti-c-Jun (1:1000, Abcam, ab40766), anti-fra1 (1:1000, Abcam, ab252421), anti-β-catenin (1:1000, Abcam, ab32572), anti-Lamin B1 (1:1000, Abcam, ab16048), anti-GAPDH (1:2000, Abcam, ab76523) and anti-β-actin (1:1000, Abcam, ab8227). Following primary antibody incubation, the membranes were washed and incubated with peroxidase-conjugated secondary antibody (1:2000, Abcam, Cambridge, UK). Subsequently, substrate development was performed using ECL chemiluminescence (Millipore, Bedford, Mass, USA).
5
Validating Cross-Reactivity of Anti-PRDX1/2 Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate cross-reactivity of anti-human PRDX1 or 2 antibodies used in the present study to canine PRDX1 or 2 proteins by western blotting, HeLa which expressed PRDX1 and 2 [9 (link), 16 (link)] and Re12 which was derived from canine HSA [27 (link)] were selected. SDS-PAGE
and western blotting was performed according to previous report [8 (link)]. Briefly, protein lysates were prepared, and electrophoresed with SDS-PAGE and then
transferred onto polyvinylidene difluoride membranes (Cytiva, Tokyo, Japan). After blocking for avoiding non-specific binding of antibodies, the membranes were incubated for 60 min at room
temperature (RT, 20–25°C) with the following primary antibodies: rabbit polyclonal antibody to human PRDX1 (1:5,000, ab228780; Abcam, Cambridge, UK), or rabbit polyclonal antibody to human
PRDX2 (1:5,000, ab109367; Abcam). Rabbit polyclonal antibody to human β-actin (1:1,000, #4967, Cell Signaling Technology, Danvers, MA, USA) was used as a loading control. The proteins were
visualized using a horse radish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000, #7074, Cell Signaling Technology) and enhanced chemiluminescence by Immobilon® Forte
Western HRP Substrate (Merck Millipore, Burlington, MA, USA). Finally, the signals were detected using a C-Digit Blot Scanner (LI-COR, Lincoln, NB, USA).
and western blotting was performed according to previous report [8 (link)]. Briefly, protein lysates were prepared, and electrophoresed with SDS-PAGE and then
transferred onto polyvinylidene difluoride membranes (Cytiva, Tokyo, Japan). After blocking for avoiding non-specific binding of antibodies, the membranes were incubated for 60 min at room
temperature (RT, 20–25°C) with the following primary antibodies: rabbit polyclonal antibody to human PRDX1 (1:5,000, ab228780; Abcam, Cambridge, UK), or rabbit polyclonal antibody to human
PRDX2 (1:5,000, ab109367; Abcam). Rabbit polyclonal antibody to human β-actin (1:1,000, #4967, Cell Signaling Technology, Danvers, MA, USA) was used as a loading control. The proteins were
visualized using a horse radish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000, #7074, Cell Signaling Technology) and enhanced chemiluminescence by Immobilon® Forte
Western HRP Substrate (Merck Millipore, Burlington, MA, USA). Finally, the signals were detected using a C-Digit Blot Scanner (LI-COR, Lincoln, NB, USA).
6
Antler Cell Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antler cells (10) were collected and lysed for 20 min using lysis buffer (RIPA lysis and 50 × Cocktail) (Servicebio, Wuhan, China). Protein concentration was examined using the BCA protein assay (Servicebio, Wuhan, China). The total protein (20 μg) was separated by SDS-polyacrylamide gel electrophoresis. Subsequently, proteins were transferred to polyvinylidene difluoride membranes and blocked with 5% skimmed milk or bovine serum albumin (BSA) powder with 0.1% Tween-20 in TBS for 2 h and incubated with primary antibody at 4 °C overnight. The following antibodies were used: anti-YAP1 (GTX129151, GeneTex, San Antonin, TX, USA, 1:1000), anti-p-YAP1 (S127) (ab76252, Abcam, Cambridge, UK, 1:2000), anti-GSK3 beta (phosphor S9) (ab75814, Abcam, 1:1000), anti-PRDX2 (ab109367, Abcam, 1:2000), anti-β-catenin (ab32572, Abcam, 1:2000), anti-Wnt5a (ab179824, Abcam, 1:2000), anti-Jak2 (3230, Cell Signaling Technology (CST) Biological reagents Company Ltd., Shanghai, China, 1:1000), anti-p-JAK2 (Try1007/1008) (3771, CST, 1:1000), anti-p-STAT3 (Tyr705) (9145, CST, 1:1000) and anti-Col2a (15943, Proteintech, Wuhan, China, 1:1000). After washing, membranes were incubated with secondary antibodies for 2 h. Lastly, the membranes were incubated with ECL chemiluminescence reagent and exposed to X-ray film for the observation of protein bands.
7
Histological Analysis of Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For histological examination, hematoxylin and eosin (H&E) staining, and Movat pentachrome staining were performed. Anti-CD31 (MAB1398Z; Millipore, Burlington, MA), anti-alpha-smooth muscle actin (anti-α-SMA; A5228; Sigma-Aldrich, St. Louis, MO; and ab5694; Abcam, Cambridge, United Kingdom), anti-CD45 (Mab114; R&D Systems, Minneapolis, MN), anti-PRDX2 (ab109367; Abcam), anti-4-hydroxynonenal (anti-4-HNE; ab46545; Abcam), anti-DNA/RNA damage antibody (anti-8-OHG) (ab62623; Abcam), and anti-mouse macrophage/monocyte antibody (anti-MOMA2) (MCA519G; Bio-Rad, Hercules, CA) were used as primary antibodies for immunostaining. After incubation with the primary antibodies, Alexa 488 and 594 (Invitrogen, Carlsbad, CA) or biotinylated secondary antibodies with 3,3′-diaminobenzidine substrate (Vector Laboratories, Burlingame, CA) were used to visualize the antigens. Furthermore, 4′,6-diamidino-2-phenylindole (DAPI) or hematoxylin was used to label the nuclei. The negative control tissues were prepared in a similar manner using IgG isotype control antibodies (Santa Cruz Biotechnology, Dallas, TX). The immunofluorescence was imaged with an LSM 510 meta confocal microscope (Carl Zeiss, Oberkochen, Germany) or a BX53 microscope (Olympus, Tokyo, Japan).
8
Protein Expression Analysis in Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were extracted in RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P‐40, 1 mM EDTA, 1 mg/ml aprotinin, 2 mg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 20 mM sodium fluoride, 0.5 mM sodium orthovanadate and centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was collected, and protein was measured using a Quant‐iT™ protein assay kit (Invitrogen). 50–100 µg of lysates were separated on a 10% SDS‐PAGE and transferred to a nitrocellulose membrane (0.45 µm, 162–0115, Bio‐Rad) using 10 mM sodium borate buffer. Transfer efficiency was determined using Ponceau S (T9026, Sigma). The membranes were blocked in 5% milk for 1 h at room temperature followed by overnight incubation at 4°C with primary antibodies: anti‐L‐plastin (1:300, MA5‐11921, Thermo Fisher), anti‐PRDX2 (1:200, AB109367, Abcam or MBS2010800, MyBiosource), anti‐PRDX4 (1:200, AB15574, Abcam), anti‐TFR‐2 (B‐6) (1:100, Santa Cruz, SC‐376278), α‐tubulin (1:5000, T9026, Sigma), anti‐TSG (1:150, Ab83, Cedarlane). The blots were washed, incubated with horseradish peroxidase‐conjugated secondary antibodies (1:1500, anti‐mouse, 170‐5047; 1:1500, anti‐rabbit, 170‐5046; Bio‐Rad) and visualized with a chemiluminescence system (Super signal West Pico; 34080, Pierce).
9
ChIRP-MS Protocol for Protein Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10–20 15 cm dishes of cells were used per ChIRP-MS experiment (100million - 500million cells depending on the cell type). Cell harvesting, lysis, disruption, and ChIRP were essentially performed as previously described [20 (link)] and follow the manufacturer’s instruction (Magna ChIRP, Millipore, USA). Final protein samples were size-separated in bis-tris SDS-PAGE gels (Invitrogen) for western blots and MS. The following antibodies were used in the followed western-blotting: anti-TLR7 (ab45371, Abcam), anti-hnRNPK (ab39975, Abcam), anti-PRDX1 (ab41906, Abcam), anti-PRDX2 (ab109367, Abcam), anti-ECM1 (ab126629, Abcam) and anti-β-actin (ab8227, Abcam). See Additional file 2 : Materials and Methods for details.
10
Western Blot Analysis of Cardiac Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We collected the left ventricular anterior wall tissue of each group of rats and extracted total protein using radioimmunoprecipitation assay (RIPA) lysate (Beyotime, Shanghai, China). A 1-mm-thick gel was used for western blot analysis. The bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China) was used to detect protein concentrations. We transferred proteins to polyvinylidene fluoride (PVDF) membranes (Roche, Basel, Switzerland) (0.2 μm) by gel electrophoresis. We then used 5% skim milk to block non-specific antigens. Prdx2 (ab109367, Abcam, Cambridge, MA, USA), TLR4 (ab22048, Abcam, Cambridge, MA, USA), p65 (ab16502, Abcam, Cambridge, MA, USA), p-p65 (ab86299, Abcam, Cambridge, MA, USA), SOD1 (ab13498, Abcam, Cambridge, MA, USA), SOD2 (ab13534, Abcam, Cambridge, MA, USA), Bax (ab32503, Abcam, Cambridge, MA, USA), and Bcl-2 (ab59348, Abcam, Cambridge, MA, USA) antibodies were used to incubate PVDF membranes at 4°C overnight. After washing the PVDF membrane with phosphate-buffered saline-tween (PBST), we used the secondary antibody dilution (ab150077, Abcam, Cambridge, MA, USA) to incubate the PVDF membrane. Finally, we used luminescent fluid to detect protein bands. β-actin was used to normalize data.
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!