Precooled lysis buffer and PMSF (Thermo Scientific, #36978, 100:1) were used to lyse SW480 and HCT116 cells for 10 min. The supernatant was incubated with 2 µg of EFTUD2 antibody (Proteintech, #10208-1-AP), c-MYC antibody (Proteintech, #10,828-1-AP), Flag antibody (Abcam, #ab236777), and V5 antibody (Abcam, #ab309485) for 1 h. Following this incubation, 40 µL of protein A/G PLUS-Agarose (Santa Cruz, #20423) beads were added. After an overnight incubation at 4 °C, the beads were washed four times with immunoprecipitation buffer. Finally, the precipitate was dissolved in 40 µL of 1× electrophoresis sample buffer, boiled for 10 min, and subsequently subjected to Western blotting using specific antibodies.
C myc antibody
Manufactured by Proteintech
Sourced in China
The C-MYC antibody is a laboratory tool used to detect and study the c-Myc protein, a transcription factor that regulates gene expression. This antibody can be used in various applications, such as Western blotting, immunohistochemistry, and immunoprecipitation, to identify and quantify the c-Myc protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
V5 antibody, by Abcam (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (1 mentions)
Flag antibody, by Abcam (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Protein a g magnetic beads, by Solarbio (1 mentions)
Pkm2 antibody, by Proteintech (1 mentions)
Fv1000, by Olympus (1 mentions)
Fish kit, by Wuhan Servicebio Technology (1 mentions)
Fasn antibody, by Proteintech (1 mentions)
Cyclin d1 antibody, by Proteintech (1 mentions)
Prrx1 antibody, by Novus Biologicals (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
Flag antibody, by Proteintech (1 mentions)
P raf, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
P mek, by Cell Signaling Technology (1 mentions)
5 protocols using c myc antibody
1
Immunoprecipitation of EFTUD2, c-MYC, Flag, and V5
2
ChIP-qPCR analysis of c-MYC binding
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The CRC cell line was cultured under standard conditions of 37 °C with 5% CO2 until it reached a density of 70–80%. To initiate cross-lining, 1% formaldehyde was applied for 10 min, and the reaction was quenched with 0.125 M glycine. Subsequently, the cells were lysed using ChIP lysis buffer supplemented with a protease inhibitor cocktail (Thermo Scientific, #87786). The lysis was sonicated, and then incubated with protein A/G magnetic beads (Solarbio, #M2400), 10 µg of c-MYC antibody (Proteintech, #10828-1-AP), or 10 µg rabbit IgG antibody (Proteintech, #30000-0-AP) as a negative control. The pulled-down and purified DNA fragments were subjected to qPCR analysis using the specified primers (Forward: 5′-cctagcaactgcgctaacag-3′, Reverse: 5′-aggaccagttccgaggtatg-3′). Relative enrichment was calculated as the amount of amplified DNA normalized to the input.
3
Multimodal Analysis of CTSLP8 Expression
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The target cells were cultured in six-well plates until the density reached 50–60%. Next, the cells were fixed using paraformaldehyde, and incubated with an immunofluorescent blocking solution for 1 h. Then, the cells were dehydrated with a gradient alcohol series, soaked in wax, and embedded in paraffin. The sections were boiled in retrieval solution for 10–15 min and allowed to cool to room temperature.
The probe for CTSLP8 was designed to be Cy3 modified, and it was synthesized by Servicebio. The detailed sequence is as follows: 5'-Cy3-GGTTTTAACCTGATCCTTCACAGGACTCAT-3'. First, the probe for CTSLP8 was detected by matched FISH kit in Servicebio. The sections were then incubated with c-Myc antibody (Proteintech; 10,828–1-AP) and PKM2 antibody (Proteintech; 15,822–1-AP). Next, the secondary antibodies of different species were added, and the sections were incubated for 50 min. Finally, the images were captured by confocal microscope (Olympus; FV1000).
The probe for CTSLP8 was designed to be Cy3 modified, and it was synthesized by Servicebio. The detailed sequence is as follows: 5'-Cy3-GGTTTTAACCTGATCCTTCACAGGACTCAT-3'. First, the probe for CTSLP8 was detected by matched FISH kit in Servicebio. The sections were then incubated with c-Myc antibody (Proteintech; 10,828–1-AP) and PKM2 antibody (Proteintech; 15,822–1-AP). Next, the secondary antibodies of different species were added, and the sections were incubated for 50 min. Finally, the images were captured by confocal microscope (Olympus; FV1000).
4
Immunohistochemical Analysis of Protein Markers
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The samples were fixed, embedded, cut into 4 μm sections and stained using a conventional immunohistochemistry procedure. Sections were incubated with FASN antibody (dilution 1:50; Proteintech), PRRX1 antibody (dilution 1:80; Novus Biologicals), cyclin D1 antibody (dilution 1:150; Proteintech) and c‐Myc antibody (dilution 1:150; Proteintech) for 2 hours, before incubated with secondary antibodies at room temperature for 30 minutes. The semiquantification of IHC results was observed and analysed by two independent pathologists. The percentage of positive cells was counted in five areas at 200× magnification per slide and was categorized into following groups: 0, <5% (−); 1, 5%‐25% (+); 2, 25%‐50% (++); 3, >50% (+++). The 0 and 1 were low expression groups, and 2 and 3 belonged to high expression groups.
5
Western Blot Analysis of Signaling Pathways
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Cells were harvested and lysed using RIPA buffer containing 1% PMSF protease inhibitor and phosphatase inhibitor. Protein concentrations were quantified using the BCA Protein Assay kit (Beyotime Biotechnology, Shanghai, China). A certain amount of protein was separated using a jacase preset gel and the proteins were electrotransferred to a PVDF membrane, blocked with skim milk for 2 h, and then incubated with primary antibodies overnight at 4 °C. The next day, the membranes were washed with a TBST solution 3 times/15 min, followed by incubation with the corresponding secondary antibodies for 2 h at room temperature. The membranes were washed again with TBST, 3 times/15 min, after which exposure was performed with ECL (Beyotime Biotechnology, Shanghai, China). Image J/FIJI was used for quantification.
The antibodies against ESM1 (Cat#ab103590), c-Met (Cat#ab216574), p-c-Met (Cat#ab68141), HIF1α (Cat#ab179483), and GAPDH (Cat# ab8245) were purchased from Abcam (Cambridge, MA, USA). The flag antibody (Cat# 20543-1-AP) and C-Myc antibody (Cat#10828-1-AP) were obtained from Proteintech (Wuhan, China). VEGFA (Cat#50661), MMP9 (Cat#13667T), p-RAF (Cat#9421), RAF (Cat#9422), p-MEK (Cat#9154), MEK (Cat#4694), p-ERK1/2 (Cat#4370), ERK1/2 (Cat#9108), p-P38 (Cat#4511), P38 (Cat#9212), p-JNK (Cat#9255), and JNK (Cat#9252) were bought from Cell Signaling Technology (Danvers, MA, USA).
The antibodies against ESM1 (Cat#ab103590), c-Met (Cat#ab216574), p-c-Met (Cat#ab68141), HIF1α (Cat#ab179483), and GAPDH (Cat# ab8245) were purchased from Abcam (Cambridge, MA, USA). The flag antibody (Cat# 20543-1-AP) and C-Myc antibody (Cat#10828-1-AP) were obtained from Proteintech (Wuhan, China). VEGFA (Cat#50661), MMP9 (Cat#13667T), p-RAF (Cat#9421), RAF (Cat#9422), p-MEK (Cat#9154), MEK (Cat#4694), p-ERK1/2 (Cat#4370), ERK1/2 (Cat#9108), p-P38 (Cat#4511), P38 (Cat#9212), p-JNK (Cat#9255), and JNK (Cat#9252) were bought from Cell Signaling Technology (Danvers, MA, USA).
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