All the chemicals and reagents were purchased from Sigma unless stated otherwise. Rabbit polyclonal anti-Pak2 antibody (Cat#: ab76293) and anti-γ-H2AX (phosphor S139) antibody (Cat#: ab81299) were obtained from Abcam (Cambridge, MA, USA); mouse monoclonal anti-α-tubulin-FITC antibody (Cat#: F2168) from Sigma (St. Louis, MO, USA); Alexa Fluor 488 goat anti-rabbit IgG (H + L) (Cat#: A11008) and CM-H2DCFDA (Cat#: C6827) from Thermo Fisher Scientific Life Technologies (Massachusetts, MA, USA); an In Situ Cell Death Detection Kit (Cat#:11684817910) was purchased from Roche (Basel, Switzerland); and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Cat#:SA00001–2), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (Cat#:SA00001–1), and GAPDH Monoclonal Antibody (Cat#:60004-1 g) were purchased from Proteintech (Wuhan, China).
Hrp conjugated affinipure goat anti mouse igg h l
Manufactured by Proteintech
Sourced in United States, China
HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) is a secondary antibody that is conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies, including both the heavy and light chains (H+L).
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Lab products found in correlation
Hrp conjugated affinipure goat anti rabbit igg h l, by Proteintech (27 mentions)
Sa00001 1, by Proteintech (4 mentions)
Gapdh monoclonal antibody, by Proteintech (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Sa00001 2, by Proteintech (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Coralite594 conjugated goat anti rabbit igg h l, by Proteintech (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
β actin, by Proteintech (2 mentions)
Goat anti rabbit igg h l, by Proteintech (2 mentions)
Protease inhibitor cocktail, by Beyotime (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti α tubulin fitc antibody, by Merck Group (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
48 protocols using hrp conjugated affinipure goat anti mouse igg h l
1
Quantifying Cellular Stress Responses
2
Regulation of Rac1, mTOR, and Cortactin
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The primary antibodies were against Rac1 (ab33186, Abcam), GAPDH (ab181602, Abcam), phosphorylated (p)-mTOR (5536T, Cell Signaling Technology, United States), mTOR (2983T, Cell Signaling Technology), ERβ (ab3576, Abcam), and cortactin (ab33333, Abcam). The secondary antibodies were horseradish peroxidase (HRP)-linked anti-rabbit immunoglobulin G (IgG) (7074S, Cell Signaling Technology), HRP-conjugated AffiniPure goat anti-mouse IgG (H++L) (SA00001-1, Proteintech, United States), and goat anti-mouse IgG H&L (Alexa Fluor® 488) (ab150113, Abcam). 17-β-Estradiol and tamoxifen was purchased from MedChemExpress to activate or modulate ER, respectively.
3
Immunofluorescence Imaging of Cellular Targets
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Reagents were rabbit secondary antibody (CoraLite594-conjugated Goat Anti- Rabbit IgG (H + L), Proteintech, Rosemont, IL, USA, article number SA00013-4), rabbit secondary antibody (CoraLite594-conjugated Donkey IgG (H + L), Proteintech, Rosemont, IL, USA, article number SA00013-8), goat secondary antibody (Fluorescein (FITC)-conjugated Affinipure Donkey Anti-Goat IgG (H + L), Proteintech, Rosemont, IL, USA, article number SA00003-3), mouse secondary antibody (horseradish peroxidase (HRP)-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) Proteintech, Rosemont, IL, USA, article number SA00001-1), and rabbit secondary antibody (HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L), Proteintech, Rosemont, IL, USA, article number SA00001-2).
Instruments included an inverted microscope (Motic, Wetzlar, Germany), fluorescence microscope, MDF-382E ultralow temperature refrigerator (Sanyo, Osaka, Japan), TONO-Pen AVIA tonometer (Medtronic SALON, USA), and HE staining kit (Bioswamp).
Instruments included an inverted microscope (Motic, Wetzlar, Germany), fluorescence microscope, MDF-382E ultralow temperature refrigerator (Sanyo, Osaka, Japan), TONO-Pen AVIA tonometer (Medtronic SALON, USA), and HE staining kit (Bioswamp).
4
NLRP3 Protein Expression Analysis
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Total cell protein was extracted using RIPA lysis buffer (cat. no. P0013B, Beyotime Institute of Biotechnology), loaded (30 µg/lane) on a 10% SDS-gel, resolved using SDS-PAGE and transferred to PVDF membranes (MilliporeSigma). The primary antibodies against NLRP3 (1:1,000; cat. no. 13158, Cell Signaling Technology, Inc.) or GAPDH (1:2,000; cat. no. TA-0A, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) were added to membranes and incubated overnight at 4°C. The secondary antibodies used were HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (1:3,000; cat. no. SA00001-2, ProteinTech Group, Inc.) and HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (1:4,000; cat. no. SA00001-1, ProteinTech Group, Inc.). The bands were visualized using a Tanon 5200 using an ECL assay and assessed using ImageJ version 1.53 (National Institutes of Health). β-actin was used as the loading control.
5
Western Blot Analysis of Protein Samples
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Harvested cells were lysed with RIPA lysis buffer (Beyotime, P0013K) and then the protease inhibitor PMSF (Sinopharm, xw020003) was added. After breaking the cells with Ultrasonic Cell Disruptor, proteins were quantified using Takara BCA Protein Assay Kit (Takara, T9300A). Protein samples (10 μg) were separated with SDS-PAGE (12%) and transferred to PDVF membrane (BIORAD). After blocking with TBST solution containing skimmed milk (Yili, 232100, 5%) or BSA (BIOFRO, BIOFROXX200010, 5%) for 1–2 h, the primary antibody was incubated at 4 °C overnight and then washed three times for 10 min each time with TBST. The secondary antibody was incubated at room temperature for 90 min. After washing the secondary antibody with TBST, membranes were incubated for 2 min by Pierce ™ ECL Western Blotting Substrate (Thermo). Membranes were imaged using the ChemiDoc ™ (BIORAD).
Primary antibodies were as follows: anti-β-actin (Proteintech, 23660-1AP, 1:10000); anti-mtTFA (Abcam, 176558, 1:7500); anti-T (CST#81694, 1:200); anti-LC3 (SANTA CRUZ, sc-398822, 1:1000). Secondary antibodies were as follows: HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Proteintech, SA00001-2, 1:2000); HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (Proteintech, SA00001-1, 1:2000).
Primary antibodies were as follows: anti-β-actin (Proteintech, 23660-1AP, 1:10000); anti-mtTFA (Abcam, 176558, 1:7500); anti-T (CST#81694, 1:200); anti-LC3 (SANTA CRUZ, sc-398822, 1:1000). Secondary antibodies were as follows: HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (Proteintech, SA00001-2, 1:2000); HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (Proteintech, SA00001-1, 1:2000).
6
Protein Extraction and Western Blot Analysis
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The radioimmunoprecipitation assay (RIPA) buffer was used to extract total protein, supplemented with 1% protease inhibitors (P8340, Sigma-Aldrich) and phosphatase inhibitors (P5726, Sigma-Aldrich). The bicinchoninic acid (BCA) assay was used to measure protein concentration. Western blot analysis was performed, as previously described (12 (link)). THBS2 (sc-136238, Santa Cruz Biotechnology), TLR4 (ab30667, Abcam), Ki67 (Proteintech Group, Inc.), HIF-1α (ab2185, Abcam), GLUT1 (ab115730, Abcam), HK2 (ab104836, Abcam), ALDOA (ab150396, Abcam), PKM2 (ab137852, Abcam), and LDHA (ab101562, Abcam) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (H+L) and HRP-conjugated AffiniPure goat anti-mouse IgG (H+L) were obtained from Proteintech Group, Inc. (Jackson).
7
Characterization of RNF183 and ERα
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Anti-RNF183 antibody (1:1,000, NBP1-74192, Novus Biologicals, Colorado, United States), anti-ERα antibody (1:1,000, ab267512, Abcam, Cambridge, United Kingdom), anti-GAPDH antibody (1:3,000, 10494-1-AP, Proteintech Group, Chicago, United States), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (1:10,000, SA00001-1 Proteintech Group, Chicago), HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:10,000, SA00001-2 Proteintech Group) were used for western blot. RNF183 (pcDNA4-myc/his-RNF183) and RNF183 without amino acids 1–60 were illustrated previously (Geng et al., 2017 (link)). The ERE-TK-Luc and the pRL-TK plasmids were constructed by the Genewiz Company (Suzhou, China).
8
Western Blotting Assay for Cas9 Detection
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The western blotting assay was performed as previously reported48 (link). Briefly, cellular extracts from HEK293T cells were prepared with lysis buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 0.5% NP-40) for 30 min at 4 °C and then denatured for 10 min at 95 °C. The cell lysates were resolved using 10% SDS-PAGE gels and transferred onto acetate cellulose membranes. For incubation, membranes were incubated with Cas9 (Beyotime Biotechnology, dilution: 1:2000, Cat:AF0123, China) or GAPDH (ABclonal, dilution: 1:10,000, Cat:AC002, USA) antibodies at 4 °C overnight followed by incubation with a secondary antibody HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (Proteintech, dilution: 1:5000, Cat:AS003, USA). Immunoreactive bands were visualized using western blotting luminal reagent (Millipore, USA) according to the manufacturer’s recommendation.
9
Cellular Senescence and Oxidative Stress Modulation
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The primary antibodies included anti-vWF (Santa Cruz, #sc-365712), anti-COL 1A1 (Proteintech, #14695-1-AP), anti-progerin (Santa Cruz, #sc-81611), anti-Lamin A/C (Cell Signaling Technology, #4777S), anti-Lamin B1 (Proteintech, #66095-1-Ig), anti-SIRT1 (Abcam, #ab110304), anti-LC3B (Abcam, #ab48394), anti-acetyl Lysine (Abcam, #ab22550), anti-Caveolin-1 (Cav-1) (Abcam, #ab32577), anti-Histone H3 (Proteintech, #17168-1-AP), and anti-GAPDH (Proteintech, #60004-1). The secondary antibodies included Cy3-labeled goat anti-mouse IgG (H+L) (Beyotime, #a0521), FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime, #a0562), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L) (Proteintech, #SA00001-1), and HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (Proteintech, #SA00001-2).
The reagents used included carbon tetrachloride (CCl4) (Sigma-Aldrich, #56-23-5), hydrogen peroxide (H2O2) (Sigma-Aldrich, #1.07298), N-Acetylcysteine (NAC) (MedChemExpress, #616-91-1), mitochondria 2,2,6,6-tetramethylpiperidinooxy (mito-TEMPO) (MedChemExpress, #1334850-99-5), 3-Methyladenine (3-MA) (Sigma-Aldrich, #S2767), rapamycin (sirolimus) (Sigma-Aldrich, #S1039), MCDB131 (Gibco, #10372019), 1640 (Gibco, #11875101), and fetal bovine serum (FBS) (Biological Industries, #04-007-1A).
The reagents used included carbon tetrachloride (CCl4) (Sigma-Aldrich, #56-23-5), hydrogen peroxide (H2O2) (Sigma-Aldrich, #1.07298), N-Acetylcysteine (NAC) (MedChemExpress, #616-91-1), mitochondria 2,2,6,6-tetramethylpiperidinooxy (mito-TEMPO) (MedChemExpress, #1334850-99-5), 3-Methyladenine (3-MA) (Sigma-Aldrich, #S2767), rapamycin (sirolimus) (Sigma-Aldrich, #S1039), MCDB131 (Gibco, #10372019), 1640 (Gibco, #11875101), and fetal bovine serum (FBS) (Biological Industries, #04-007-1A).
10
Epitope Mapping of Gc Antibodies
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To map the epitopes of the Gc antibodies, four truncated Gc fragments (aa 1–142, 132–262, 252–375, and 365–521) of CCHFV-IbAr10200 were PCR-amplified and cloned into the pET32a prokaryotic expression plasmid (Novagen). Protein samples were subjected to SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. Subsequently, the membrane was blocked with 5% non-fat dried milk for 1 h at 25°C, followed by incubation with Gc8, Gc13, or Gc35 for 2 h at 1:5,000 dilution. After washing with TBS-T (20 mM Tris-base, 150 mM NaCl, 0.05% Tween-20, pH 7.5), the blots were incubated with HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, 1:5,000 dilution in PBS-T) for 1 h. Final signals were detected using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific) and photographed under MicroChemi (DNR Bio-Imaging Systems, DNR).
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