Protein extraction was performed as described in Thomas et al. (2015) [45 (link)] using approximately 150 µL of ground leaf tissue of N. benthamiana. The protein samples were mixed with a 2× protein gel loading buffer (NuPAGETM LDS Sample Buffer (4X) and NuPAGETM Sample Reducing Agent (10X)) and incubated at 95 °C for 10 min. Next, the samples were separated on a Bis–Tris gel as described in Huwa et al. (2021) [13 (link)], and the proteins were transferred onto nitrocellulose membranes for immunodetection using Anti-HA (rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA) and Anti-cMyc antibodies (mouse, 1:2000, obtained from U. Commandeur, RWTH Aachen) as the primary antibodies. Anti-rabbit-HRP (goat, 1:2000, Cell Signaling Technology, Danvers, MA, USA) and Anti-mouse-HRP (horse, 1:2000, Cell Signaling Technology, Danvers, MA, USA) were used as the secondary antibodies.
Anti c myc antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-c-Myc antibody is a laboratory reagent used to detect and study the c-Myc protein. c-Myc is a transcription factor that plays a critical role in cell growth, proliferation, and apoptosis. The antibody is designed to specifically bind to the c-Myc protein, allowing researchers to study its expression and localization in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin antibody, by Merck Group (4 mentions)
Anti ppp2r5a, by Abcam (2 mentions)
Anti p c myc s62 antibodies, by Cell Signaling Technology (2 mentions)
Mouse monoclonal α tubulin fitc antibody, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Gapdh antibody, by Cell Signaling Technology (2 mentions)
Anti jund antibody, by Cell Signaling Technology (2 mentions)
Anti fosb antibody, by Cell Signaling Technology (2 mentions)
Chip assay kit, by Thermo Fisher Scientific (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (1 mentions)
Colcemid, by Merck Group (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
7 hydroxystaurosporine ucn 01, by Merck Group (1 mentions)
Anti caspase 8, by Abcam (1 mentions)
Anti rad51, by Cell Signaling Technology (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
30 protocols using anti c myc antibody
1
Protein Extraction and Western Blotting
2
Western Blot Antibody Cocktail
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Anti-γ-H2AX, anti-cleaved caspase3, anti-cleaved caspase9, anti-PARP, anti-p53, anti-RAD51, anti-phospho-STAT5A (Tyr 694), anti-STAT5A, and anti-c-myc antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-caspase2, anti-caspase8, and anti-caspase10 were purchased from Abcam (Cambridge, UK). Anti-phospho-STAT5A (Ser 726) and anti-phospho-STAT5A (Ser 780) were purchased from OriGene Technologies (Rockville, MD, USA). The anti-β-actin antibody was obtained from Sigma-Aldrich (Saint Louis, MO, USA), and peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies were purchased from ZSGQ-BIO Company (Beijing, China). Anti-γ-H2AX (Ser139), conjugated with fluorescein isothiocyanate (FITC), was purchased from BD Company (Franklin Lakes, NJ, USA). 7-Hydroxystaurosporine (UCN-01) and colcemid were purchased from Sigma-Aldrich (Saint Louis, MO, USA).
3
UHRF1 and c-Myc Immunoprecipitation
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The cells were lysed in IP Lysis buffer (87788, Thermo Scientific) along with a protease inhibitor cocktail (Roche Applied Science, Upper Bavaria, Germany) at 4 °C for 30 min. Thereafter, the cell extract was centrifuged at 14,000 rpm for 15 min at 4 °C, and 5% of the cell extract was kept for input. To conjugate the primary antibody, the cell extracts were incubated with 2 µg of the anti-UHRF1 (Santa Cruz), anti-MYC (Cell Signaling), or rabbit IgG antibody and Dynabeads protein G (Thermo Fisher Scientific) for overnight incubation at 4 °C. The antibody–antigen complex was washed three times with PBS. Laemmli buffer (Bio-Rad, Hercules, CA, USA) was subsequently added to elute the precipitated proteins, followed by Western blotting using anti-UHRF1 (sc-373750, Santa Cruz) and anti-c-Myc antibodies (5605S, Cell Signaling). The immunoblot conditions were the same as described for the siRNA-treated samples.
4
Western Blot Analysis of Stem Cell Markers
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Cells were lysed by RIPA buffer and centrifuged (12,000 g × 30 min) to collect total protein in the supernatant, which were mixed with 5× SDS loading buffer (P0015, Beyotime, Shanghai, China) and heated to 100°C (10 min) for denaturation. After SDS-PAGE and transfer to the polyvinylidene difluoride (PVDF) membrane, the membranes were blocked by 5% skim milk solution and incubated with the corresponding primary antibodies at 4°C for a night. After three times wash, the membranes were incubated with secondary antibody and then washed for another three times, followed by chemiluminescent exposure of the blot with NcmECL Ultra (P10100, NCM Biotech, Suzhou, China). The primary antibodies were as follows: Anti-CD44 antibodies (15675-1-AP, Proteintech, Chicago, The United States), Anti-CD133 antibodies (18470-1-AP, Proteintech), Anti-SOX2 antibodies (#3579, Cell Signaling Technology, Massachusetts, The United States), Anti-C-MYC antibodies (#5605, Cell Signaling Technology), Anti-GAPDH antibodies (GB11002, Servicebio, Wuhan, China).
5
Protein Analysis of Gastric Cancer Cells
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Gastric cancer cells with indicated treatment were rinsed with cold PBS before treated with RIPA lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EGTA, 1% NP40, 0.25% SDS, pH 7.4) containing protease and phosphatase inhibitors. Cell pellets were incubated for 30 min on ice to homogenize fully and then total proteins in the supernatant of cell lysates were collected by centrifuging at 4°C at 12,000 rpm for 10 min.
Total protein concentrations were measured by the BCA protein assay kit (Pierce, USA) before experiment. Then, standard western blotting assays (SDS-PAGE) were used to analyze protein expression. The antibodies used for western blotting analysis in this study included: anti-PPP2R5A (Abcam, ab89621, 1:2,000 dilution), anti-c-Myc antibodies (Cell Signaling Technology, #5605, 1:1,000 dilution), anti-p-c-Myc (S62) antibodies (Cell Signaling Technology, #13748,1:1,000 dilution), anti-β-actin antibody (Sigma, A2228, 1:5,000 dilution). After incubation with corresponding species-speci c secondary HRP-conjugated antibodies, the target protein bands were visualized by an ECL imaging system.
Total protein concentrations were measured by the BCA protein assay kit (Pierce, USA) before experiment. Then, standard western blotting assays (SDS-PAGE) were used to analyze protein expression. The antibodies used for western blotting analysis in this study included: anti-PPP2R5A (Abcam, ab89621, 1:2,000 dilution), anti-c-Myc antibodies (Cell Signaling Technology, #5605, 1:1,000 dilution), anti-p-c-Myc (S62) antibodies (Cell Signaling Technology, #13748,1:1,000 dilution), anti-β-actin antibody (Sigma, A2228, 1:5,000 dilution). After incubation with corresponding species-speci c secondary HRP-conjugated antibodies, the target protein bands were visualized by an ECL imaging system.
6
Protein Analysis of Gastric Cancer Cells
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Gastric cancer cells with indicated treatment were rinsed with cold PBS before treated with RIPA lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EGTA, 1% NP40, 0.25% SDS, pH 7.4) containing protease and phosphatase inhibitors. Cell pellets were incubated for 30 min on ice to homogenize fully and then total proteins in the supernatant of cell lysates were collected by centrifuging at 4°C at 12,000 rpm for 10 min.
Total protein concentrations were measured by the BCA protein assay kit (Pierce, USA) before experiment. Then, standard western blotting assays (SDS-PAGE) were used to analyze protein expression. The antibodies used for western blotting analysis in this study included: anti-PPP2R5A (Abcam, ab89621, 1:2,000 dilution), anti-c-Myc antibodies (Cell Signaling Technology, #5605, 1:1,000 dilution), anti-p-c-Myc (S62) antibodies (Cell Signaling Technology, #13748,1:1,000 dilution), anti-β-actin antibody (Sigma, A2228, 1:5,000 dilution). After incubation with corresponding species-speci c secondary HRP-conjugated antibodies, the target protein bands were visualized by an ECL imaging system.
Total protein concentrations were measured by the BCA protein assay kit (Pierce, USA) before experiment. Then, standard western blotting assays (SDS-PAGE) were used to analyze protein expression. The antibodies used for western blotting analysis in this study included: anti-PPP2R5A (Abcam, ab89621, 1:2,000 dilution), anti-c-Myc antibodies (Cell Signaling Technology, #5605, 1:1,000 dilution), anti-p-c-Myc (S62) antibodies (Cell Signaling Technology, #13748,1:1,000 dilution), anti-β-actin antibody (Sigma, A2228, 1:5,000 dilution). After incubation with corresponding species-speci c secondary HRP-conjugated antibodies, the target protein bands were visualized by an ECL imaging system.
7
Antibody Reagents for Protein Detection
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Mouse monoclonal anti-NET1 antibodies (Cat# 28180-1-AP) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-RAC1 antibodies (Cat#: 24072-1-AP), mouse polyclonal anti-HACE1 antibodies (Cat# 24104-1-AP), and mouse monoclonal anti-GAPDH antibodies (Cat#: 60004-1-Ig) were purchased from Proteintech (Wuhan, China). Mouse monoclonal α-tubulin-FITC antibody (Cat# F2168) was from Sigma (St. Louis, MO, USA); and anti-c-MYC-antibody (Cat# 2278 S), HRP-conjugated rabbit (Cat #7074S), and mouse (Cat# 7076 S) secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA). We purchased Fluor 555 (Cat# A-11,001) and 488 (Cat# A-21,429) conjugated anti-mouse and anti-rabbit secondary antibodies from Invitrogen (USA).
8
ChIP-qPCR Analysis of VEGFA Promoter
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Stably transfected HEK293 or HUVEC cells were fixed with formaldehyde (1% final concentration) for 10 min at room temperature. The reaction was stopped by adding 125 mM of glycine. ChIP assays were performed according to the protocol of ChIP-IT Express Enzymatic kit (Active Motif) with affinity purified polyclonal anti-SerRS antibody (custom-made), anti-c-Myc antibody (Cell Signaling Technology, #9402), and anti-HIF-1α antibody (Cell Signaling Technology, #36169). After 3 washes, ChIPed DNA was analyzed on the StepOnePlus Real-Time PCR system using SYBR Select Master Mix (Applied Biosystems). The primer set (forward: 5′-GGGCGGATGGGTAATTTTCA-3′, reverse: 5′- CTGCGGACGCCCAGTGAA- 3′) targeting the VEGFA promoter was used.
9
Rhein-induced Protein Expression in HaCaT Cells
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After co-culture with 50 μM rhein for 24 h, HaCaT cells were washed with sterilized phosphate buffered saline (PBS) and harvested from 6-well plates using 50 μL RIPA lysis buffer (Beyotime, China) per well. And BCA assay was performed to measure the protein concentration with BCA protein analysis kit (Thermo Fisher Scientific Inc, U.S.). For each blotting process, a total amount of 10 μg protein per sample was separated and transferred to a polyvinylidene fluoride membrane. The membranes were then incubated with the following primary antibodies overnight at 4 °C: anti-c-myc antibody (Cell Signaling Technology, 5605S, 1:1000), anti-FosB antibody (Cell Signaling Technology, 2251S, 1:1000), anti-JunD antibody (Cell Signaling Technology, 5000S, 1:1000), and GAPDH antibody (Cell Signaling Technology, 2118S, 1:5000). The membranes were then incubated with appropriate secondary antibodies (MedChemExpress, 7074S, 1:10,000) for 1 h at room temperature.
10
Inducible ERG Expression in LNCaP Cells
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LNCaP cell line was transduced with an inducible lentiviral ERG construct (LNCaP-lentivirus TMPRESS2:ERG3 inducible) to establish stable doxycycline-inducible ERG expressing LnTE3 cell line [2 (link), 16 (link)]. The cell lines were cultured in RPMI 1640, supplemented with 10% Tet System Approved Fetal Bovine Serum (Clontech Laboratories, Inc. Mountain View, CA, USA) and puromycin (Sigma, St. Louis, MO, USA) with or without doxycycline (Dox, 1 μg/ml) as per requirements and characterized as described [2 (link), 16 (link)]. Antibodies used were as follows: anti-GAPDH (Millipore MAB374), anti-ERG (Abcam ab92513), anti- p21Waf1/Cip1, anti-E2F1 and anti- c-Myc Antibody (Cell Signaling 2946, 3742 and 9402, respectively), anti-p53 DO1 (Santa Cruz biotech, sc126), and anti-NKX3.1 (Biocare Medical SKU 422).
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