Proteins were separated on 4–12% Bis-Tris reducing polyacrylamide gels (Life Technologies) and transferred to nitrocellulose using iBlot (Thermo Fisher Scientific). Membranes were blocked for 1 h with 5% Skim Milk in PBS + 0.1% Tween-20. Monoclonal mouse antibodies against HA (12CA5), FLAG 9H1 and FLAG (M2) were used at 1:1000. Polyclonal Antibodies against RhopH2, RhopH3 and Clag3 were generated in rabbits (GenScript) against the following antigens: RhopH3 T731-Y829, RhopH2 L20-S1378 and Clag3 K1277-H1417. Rabbit polyclonal antibodies were used at 1:500–1:1000 dilution. The following secondary HRP-conjugated antibodies were used: goat a-rat (Southern Biotech, 3030-05), goat α-mouse (Merck Millipore, AP124P), goat a-rabbit (Merck Millipore, AP187P).
Ap124p
Manufactured by Merck Group
Sourced in United States, China
The AP124P is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of this product is to provide a reliable and precise platform for various laboratory applications. Detailed technical specifications and intended use are not available for this product.
Automatically generated - may contain errors
Lab products found in correlation
Ap132p, by Merck Group (22 mentions)
β actin, by Merck Group (5 mentions)
Zb 2301, by ZSGB-BIO (3 mentions)
Bca protein assay kit, by Keygen Biotech (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Mca1396, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Bm0627, by Boster Bio (2 mentions)
Ecl western blot kit, by Merck Group (2 mentions)
Goat anti mouse igg, by Merck Group (2 mentions)
Goat anti rabbit igg, by ZSGB-BIO (2 mentions)
Mouse anti β actin, by Cell Signaling Technology (2 mentions)
Minichemi, by Thermo Fisher Scientific (2 mentions)
Abs16, by Merck Group (2 mentions)
Ab51520, by Merck Group (2 mentions)
Pab12474, by Abcam (2 mentions)
P8340, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Goat a rat, by Southern Biotech (1 mentions)
43 protocols using ap124p
1
Western Blot Analysis of Malaria Proteins
2
Western Blot Protein Analysis
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Total tissue and cell proteins were extracted following the instructions of RIPA lysate (Beyotime, Shanghai, China). Western blot was performed using 40‐50 μg of protein lysates. Proteins were separated using 12.5% SDS‐PAGE and transferred onto PVDF membranes (AP124P, Merck, Germany). The remaining steps were performed strictly in accordance with the instructions of each antibody. Protein bands were detected using enhanced chemiluminescence (32109, Thermo Scientific, Waltham, MA, USA). The antibodies used in the study, including origin and dilution, are listed in Table S2 . Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as a reference.
3
Western Blot Analysis of HYAL2 and SOD3
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Protein was isolated from lung tissue in T-PER buffer and quantified with BCA assay as described above. Protein from cultured cells was isolated in RIPA lysis buffer (Thermo-Fisher). Under reducing conditions, protein was loaded onto a 4–12% precast gradient PAGE gel (Criterion Bis-Tris HCl XT, BioRad). Samples were electrophoresed at 200 V for 55 minutes. The protein was then transferred onto PVDF as described before. The membrane was blocked in 5% milk/TBST for 1 hour and probed with rabbit anti-mouse polyclonal HYAL2 antibody diluted to 1:500 (Abcam #68608; Cambridge, UK) overnight at 4 °C. This antibody has been used to detect HYAL2 in human aortic valve interstitial cells cultured in hypoxia34 (link). SOD3 from HPASMCs was probed with mouse-anti-human monoclonal antibody (sc-376948, Santa Cruz Biotech; Dallas, TX). Loading controls were either mouse monoclonal β-actin (Sigma #A2228, used at 1:10 K) or rabbit-anti-human polyclonal Vinculin (Cell Signaling Technologies #4650, used at 1:1 K). The membranes were then probed with mouse polyclonal IgG-HRP 2° antibody (EMD Millipore #AP124P, used at 1:10 K) or goat anti-rabbit 2° antibody at 1:10 K for 1 hour. Blots were developed with Clarity ECL, imaged under trans-UV, and quantitated by relative densitometry as previously described.
4
Quantifying Fungal Protein Expression
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Total protein extraction was performed as described previously (111 (link)), except for the lysis step. Frozen mycelia were lysed in 500 μl ice-cold denaturing buffer (10 mM Tris-HCl, pH 8.0, 25 mM NH4 acetate, 1 mM EDTA, 10% trichloroacetic acid) with an ∼100-μl volume of silica beads through six cycles of 3 min of beating using a Bullet Blender. Samples were allowed to cool on ice for at least 3 min between each cycle. Total protein concentration was measured using the Bio-Rad DC protein assay kit (5000111). The same amounts of proteins (50 μg) from different samples were subjected to SDS-PAGE and Western blotting. The anti-HA (Santa Cruz sc-7392 and Immunoway YM3003; 1:2,000 dilution) and anti-histone H3 (ab1791; 1:5,000 dilution; Abcam) primary antibodies and the horseradish peroxidase (HRP)-conjugated anti-rabbit (AP132P; 1:5,000 dilution; Merck Millipore) and HRP-conjugated anti-mouse (AP124P; 1:5,000 dilution; Merck Millipore) secondary antibodies were used.
5
Nanobody Binding Assay on Recombinant Proteins
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In total, 96-well flat-bottomed MaxiSorp plates were coated with 65 nM of recombinant protein as indicated in 50 μl of PBS at room temperature (RT) for 1 h. All washes were done three times using PBS and 0.1% Tween (DPBS-T) and all incubations were performed for 1 h at RT. Coated plates were washed and blocked by incubation with 10% skim milk solution. Plates were washed and then incubated with 65 nM of nanobodies. The plates were washed and incubated with mouse anti-His (Bio-Rad MCA-1396; 1 : 1000) followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (MerckMillipore AP124P, 1 : 1000). After a final wash, 50 μl of azino-bis-3-ethylbenthiazoline-6-sulfonic acid (ABTS liquid substrate; Sigma) was added and incubated in the dark at RT and 50 μl of 1% SDS was used to stop the reaction. Absorbance was read at 405 nm and all samples were done in duplicate.
6
Antibodies and HDAC Inhibitors in Cell Study
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The following primary antibodies were used in this study: Myc monoclonal antibody (M4439, Sigma-Aldrich), β-actin (A5441, Sigma-Aldrich), Flag (F3165, Sigma-Aldrich), HDAC1 (10197-1-AP, Proteintech), TARDBP (190782-2-AP, Proteintech), Acetylated-Lysine antibody (9441, Cell Signaling), GFP (33-260, ThermoFisher Scientific), caspase-3 (♯9662, Cell Signaling Technology), PARP (♯9542, Cell Signaling Technology), LC3B (♯2775, 2Cell Signaling Technology), anti-rabbit peroxidase-conjugated secondary antibody (AP132P EMD Millipore) and anti-mouse peroxidase-conjugated secondary antibody (AP124P EMD Millipore); anti-rabbit, anti-mouse Alexa 488 (A-11001, Life Technologies) or 647-conjugated secondary antibody (A-21244, Life Technologies). All antibodies were used at the dilution recommended by the manufacturer’s instructions.
The following HDACis were used in this study: Sodium phenil butyrate (SML0309, Sigma-Aldrich), Trichostatin A (T8552, Sigma-Aldrich), Sodium butyrate (B5887, Sigma-Aldrich), Valproic acid sodium salt (P4543, Sigma-Aldrich).
The following HDACis were used in this study: Sodium phenil butyrate (SML0309, Sigma-Aldrich), Trichostatin A (T8552, Sigma-Aldrich), Sodium butyrate (B5887, Sigma-Aldrich), Valproic acid sodium salt (P4543, Sigma-Aldrich).
7
Investigating RBM10 Regulation in A549 and H1299 Cells
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A549 and H1299 cells were transfected with pcDNA3.1-RBM10 wild type or mutated cDNA or vector-only for 48 h. Total cellular protein was then extracted using the radioimmunoprecipitation assay lysis buffer and protein concentrations were measured using bicinchoninic acid (BCA) kit (CWBiotech, Beijing, China). Equal amounts of protein samples (20 µg each) were heated at 100°C for 10 min and then separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidenedifluoride (PVDF) membrane (EMD Millipore). The membranes were incubated in 5% bovine serum albumin (BSA) for 2 h at the room temperature and then incubated with primary antibodies at a dilution of 1:1,000 against Numb (1:1,000; cat. no: 18701-1-AP), Notch-1 (1:1,000; cat. no: 10062-2-AP), Fas (1:1,000; cat. no: 13098-1-AP), E-cadherin (1:1,000; cat. no: 20874-1-AP), and CyclinD1 (1:1,000; cat. no: 60186-1-Ig; all from ProteinTech Group, Inc., Chicago, IL, USA) at 4°C overnight. On the next day, the membranes were washed with Tris-based saline-Tween 20 solution (TBST) three times and then incubated with a secondary antibody at a dilution of 1:1,000 (1:1,000; cat. no: AP124P; EMD Millipore) for 1 h at the room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL)-Plus western blotting detection reagents (EMD Millipore).
8
Quantitative ELISA for Nanobody Screening
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The 96-well flat-bottomed MaxiSorp plates were coated with 125 nM of recombinant protein, as indicated, in 50 μL DPBS at room temperature for 1 h. All washes were done three times using PBS and 0.1% Tween, and all incubations were performed for 1 h at room temperature. Coated plates were washed and blocked by incubation with 10% skim milk solution. Plates were washed and then incubated with 125 nM of nanobodies. The plates were washed and incubated with mouse anti-His (Bio-Rad MCA-1396; 1:1,000) followed by horseradish peroxidase–conjugated goat anti-mouse secondary antibody (MerckMillipore AP124P, 1:1,000). After a final wash, 50 μL azino-bis-3-ethylbenthiazoline-6-sulfonic acid (liquid substrate; Sigma) was added and incubated in the dark at room temperature, and 50 μL 1% SDS was used to stop the reaction. Absorbance was read at 405 nm, and all samples were done in duplicate.
9
Antibody Immunoblot and Immunofluorescence Protocol
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Antibodies against the following proteins were obtained from commercial sources and used for immunoblots: anti-flag-tag (F3165, Sigma), anti-V5-tag (R960-25, Thermo Fisher Scientific), anti-puromycin (MABE343, Merck Millipore), anti-GAPDH (MAB374, Merck Millipore), anti-phospho-eIF2α Ser51 (3398, CST), anti-TRIM21 (92043, CST), anti-PKR (12297, CST), anti-Myc tag (2276, CST), anti-HA tag (ab236632, Abcam), anti-TRIM21 (ab91423, Abcam), anti-phospho-PKR T451 (ab81303, Abcam), and anti-IRF1 (8478, CST). Anti-interferon alpha/beta receptor 1 (ab45172, Abcam), anti-STAT2 (72604, CST), anti-PP1α (ab137512, Abcam), anti-ISG15 (ab285367, Abcam), anti-GST mouse monoclonal antibody (HT601-01, Transgen), β-actin (A5541, Sigma), and goat anti-mouse IgG (HRP-linked) (AP124P, Merck Millipore) were also used. The following antibodies were obtained from commercial sources and used for immunofluorescence staining: anti-puromycin (MABE343, Merck Millipore) and donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody conjugated with Alexa Fluor 594 (A-21207, Thermo Fisher Scientific). The mouse monoclonal anti-HCV core antibody was a gift from Chen Liu.
10
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cell lines using mammalian protein extraction reagent (MPER; 50 nM Tris-HCl, 200 mM NaCl, 0.25% Triton 100X, and 10% glycerol) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, PIA32961). Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, 500-0006). Twenty micrograms of total protein extract were separated in precast 4–15% gradient Tris-glycine SDS-polyacrylamide gels (Mini-PROTEAN® TGX™ Precast Gels, Bio-Rad, 456-1086) and transferred onto Trans-Blot Turbo Mini 0.2 µm nitrocellulose membranes (Bio-Rad, 170-4159). Membranes were blocked with 5% milk in PBS-Tween for 1 h and probed with primary antibodies overnight at 4 °C with agitation. Primary antibodies were detected with peroxidase-conjugated secondary antibodies Goat anti-mouse (Millipore, AP124P, 1:4000 dilution), Goat anti-rabbit (Millipore, AP156P, 1:10,000 dilution), and Rabbit anti-goat (Millipore, AP106P, 1/4000 dilution) and enhanced with chemiluminescence (Millipore, RPN2232) detected using the ChemiDoc MP Imaging System (Bio-Rad). Actin was used as a loading protein control. Each experiment was repeated three times. Image J was used to quantify western blot.
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