Phototope horseradish peroxidase western blot detection kit

Manufactured by Thermo Fisher Scientific

The Phototope Horseradish Peroxidase Western Blot Detection kit is a laboratory product designed for the detection and visualization of target proteins in Western blot analyses. It utilizes horseradish peroxidase (HRP) enzyme-based chemiluminescence for protein detection.

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Lab products found in correlation

Pvdf membrane, by Merck Group (5 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) Ab195352, by Abcam (1 mentions) Ab71973, by Abcam (1 mentions) Ab126811, by Abcam (1 mentions) Ab183758, by Abcam (1 mentions) Ab32359, by Abcam (1 mentions) Ab31218, by Abcam (1 mentions) F1804, by Santa Cruz Biotechnology (1 mentions) Srcin1, by Cell Signaling Technology (1 mentions) β actin, by Proteintech (1 mentions) Phos tag acrylamide, by Apexbio (1 mentions) Antibody against, by Merck Group (1 mentions) Flag tag (flag), by Merck Group (1 mentions) Normal mouse igg sc 2025, by Santa Cruz Biotechnology (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Cstf 77, by Fortis Life Sciences (1 mentions)

7 protocols using phototope horseradish peroxidase western blot detection kit

Western Blot Analysis of Protein Targets

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Protein extracts from cells or immunoprecipitation samples were prepared using detergent-containing lysis buffer. Total protein (50 µg) was subjected to SDS-PAGE and transferred to PVDF membrane (Millipore). Antibody against METTL3 (ab195352), PHLPP1 (ab71277), PHLPP2 (ab71973), AKT1/2/3 (ab126811), p-AKT1/2/3 (p-S472 + S473 + S474; ab183758), p70S6K (ab32359), p-p70S6K (p-T389; ab126818), NKAP (ab121121), IF4A2 (ab31218), SLU7 (ab151462), PCMD1 (ab121858), PLXA4 (ab127892), CENPJ (ab26052), FLIP1 (ab205925), or DROSHA (ab12286) was from Abcam. Antibody against NFIC (sc-74444), FLAG tag (F1804), 6 × His tag (SAB2702218) or β-ACTIN (66009-1-Ig) was from Santa Cruz Biotechnology, Sigma, or Proteintech, respectively. Membranes were incubated overnight at 4 °C with primary antibody and visualized with a Phototope Horseradish Peroxidase Western Blot Detection kit (Thermo Fisher).

Zhang J., Bai R., Li M., Ye H., Wu C., Wang C., Li S., Tan L., Mai D., Li G., Pan L., Zheng Y., Su J., Ye Y., Fu Z., Zheng S., Zuo Z., Liu Z., Zhao Q., Che X., Xie D., Jia W., Zeng M.S., Tan W., Chen R., Xu R.H., Zheng J, & Lin D. (2019). Excessive miR-25-3p maturation via N6-methyladenosine stimulated by cigarette smoke promotes pancreatic cancer progression. Nature Communications, 10, 1858.

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SRCIN1 Expression Analysis by Western Blot

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Consistent with previous experimental procedures, Western blot analysis was conducted to assess SRCIN1 expression (10 (link)). Protein was extracted from the cell lines, and the immunoprecipitation samples were prepared using detergent-containing lysis buffer. Total protein (60 μg) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were incubated with primary antibodies against SRCIN1 (Cell Signaling Technology, dilution: 1:1,000) and β-actin (Proteintech, dilution: 1:1,000) overnight at 4°C, and the proteins were detected with a Phototope horseradish peroxidase Western blot detection kit (Thermo Fisher).

Dong J., Li B., Lin D., Lu D., Liu C., Lu X., Tang X., Li L., Zhu D., Liu J., Qiu X., Tian L, & Zhou Q. (2020). LincRNA00494 Suppresses Non-small Cell Lung Cancer Cell Proliferation by Regulating SRCIN1 Expression as a ceRNA. Frontiers in Oncology, 10, 79.

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Western Blot Analysis of ESCC Proteins

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Protein from ESCC tissues or cells was extracted using detergent-containing lysis buffer. Total protein (30 μg) was subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica). The membranes were incubated overnight at 4°C with a specific antibody and visualized with a Phototope Horseradish Peroxidase Western Blot Detection kit (Thermo Fisher Scientific). Detailed information about the antibody against target proteins is shown in Table S8.

Li R., Zeng L., Zhao H., Deng J., Pan L., Zhang S., Wu G., Ye Y., Zhang J., Su J., Zheng Y., Deng S., Bai R., Zhuang L., Li M., Zuo Z., Lin D., Zheng J, & Huang X. (2022). ATXN2-mediated translation of TNFR1 promotes esophageal squamous cell carcinoma via m6A-dependent manner. Molecular Therapy, 30(3), 1089-1103.

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Immunoblot Analysis of SMAD4, AMPK, and HNF4G

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Protein extracts from cells were prepared using detergent-containing lysis buffer. Total protein (20 µg) was subjected to SDS-PAGE and transferred to PVDF membrane (Millipore). Antibody against SMAD4 (#46535), AMPK (#5831), p-AMPK (Thr172, #2535), or ubiquitin (#3933) was from Cell Signaling. Antibody against HNF4G (#25801-1-AP) or β-ACTIN (#TA-09) was from Proteintech and ZSGB-BIO. Membranes were incubated overnight at 4 °C with primary antibody and visualized with a Phototope Horseradish Peroxidase Western Blot Detection kit (Thermo Fisher). HNF4G phosphorylation assay was performed using Phos-tag Acrylamide (#F4002, APExBIO) according to the instructions.

Wang C., Zhang T., Liao Q., Dai M., Guo J., Yang X., Tan W., Lin D., Wu C, & Zhao Y. (2020). Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation. Protein & Cell, 12(2), 128-144.

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Proteomic Analysis of PDAC Tissues

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Total protein extract from PDAC tissues or cells was prepared using a detergent-containing lysis buffer. For cytoplasmic and nuclear fractionation, lysis was obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo) following the manufacturer’s instructions. Protein sample (50 μg) was subjected to SDS-PAGE and transferred to the PVDF membrane (Millipore). Antibody against CSTF2 (ab200837), CSTF2T (ab138486), METTL3 (ab195352), METTL14 (ab252562), WTAP (ab195380), FTO (ab126605), ALKBH5 (ab195377), IGF2BP2 (ab128175), WNT7B (ab227607), RNA polymerase II C-terminal domain (CTD) Ser2 (ab193468) or β-ACTIN (ab8227) was from Abcam. Antibody against RNA polymerase II C-terminal domain (CTD) (#05-623) and were from Millipore. Antibody against U2AF2 (68166-1-Ig), CAPRIN1 (15112-1-AP), RBM15 (10587-1-AP), RBM15B (67506-1-Ig), Lamin B1 (12987-1-AP), GAPDH (60004-1-lg), AFF4 (14662-1-AP) or CENPF (28568-1-AP) were from Proteintech. Antibody against BUD13 (A303-321A-1) and AFF1 (A302-345A-1) were from Invitrogen and Antibody against NTSR1 (sc-374492) was from Santa Cruz Bio. The membrane was incubated overnight at 4 °C with primary antibody and visualized with a Phototope Horseradish Peroxidase Western Blot Detection kit (Thermo Fisher).

Zheng Y., Li X., Deng S., Zhao H., Ye Y., Zhang S., Huang X., Bai R., Zhuang L., Zhou Q., Li M., Su J., Li R., Bao X., Zeng L., Chen R., Zheng J., Lin D., He C., Zhang J, & Zuo Z. (2023). CSTF2 mediated mRNA N6-methyladenosine modification drives pancreatic ductal adenocarcinoma m6A subtypes. Nature Communications, 14, 6334.

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Western Blotting of Circularized Proteins

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Western blotting was performed as per standard protocols. Proteins were separated on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Membranes were incubated overnight at 4 °C with primary antibodies (FLAG tag and anti-CORO1C47aa) and visualized with a Phototope Horseradish Peroxidase Western Blot Detection kit (Thermo Fisher). A polyclonal antibody against the CORO1C-47aa peptide produced by circ-0000437 was made by ChinaPeptides Co, Ltd. The other primary antibodies: ARNT (A-3: sc-17811, Santa Cruz), TACC3 (ab138262, Abcam), VEGFA (ab1316, Abcam), VEGFR2 (2479S, CST), FLAG tag (F1804, Sigma) and HA tag (clone 12CA5, Santa Cruz). Normal mouse IgG (sc-2025) was from Santa Cruz. Antibody against beta-ACTIN (66009-1-Ig) was from Proteintech. Anti-mouse IgG (CST 7056) and Anti-rabbit IgG (CST 7054) were used, respectively.

Li F., Cai Y., Deng S., Yang L., Liu N., Chang X., Jing L., Zhou Y, & Li H. (2021). A peptide CORO1C-47aa encoded by the circular noncoding RNA circ-0000437 functions as a negative regulator in endometrium tumor angiogenesis. The Journal of Biological Chemistry, 297(5), 101182.

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Western Blot Analysis of Protein Targets

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Protein extracts from cells or tissue samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). Antibody against BID (Cat# 2002, RRID: AB_10692485) was from Cell Signaling Technology. Antibody against ZFP36L2 (Cat# sc-365908, RRID: AB_10847357) was from Santa Cruz Biotechnology. Antibody against CstF64 (Cat# A301-092A, RRID: AB_873014), CstF77 (Cat# A301-096A, RRID: AB_873019), and CPSF100 (Cat# A301-582A, RRID: AB_1078863) was from Bethyl Laboratories. Membranes were incubated overnight at 4 C with primary antibody and visualized with a phototope-horseradish peroxidase Western Blot Detection kit (Thermo Fisher Scientific).

Lin A., Ji P., Niu X., Zhao X., Chen Y., Liu W., Liu Y., Fan W., Sun Y., Miao C., Zhang S., Tan W., Lin D., Wagner E.J, & Wu C. (2021). CstF64-Induced Shortening of the BID 3'UTR Promotes Esophageal Squamous Cell Carcinoma Progression by Disrupting ceRNA Cross-talk with ZFP36L2. Cancer research, 81(22).

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