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Ab57154

Manufactured by Abcam
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Ab57154 is a laboratory reagent. It is a highly specific antibody that can be used to detect a particular target protein in biological samples. The core function of this product is to facilitate the identification and quantification of the target protein through immunological techniques.

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Lab products found in correlation

Prs3959, by Abcam (4 mentions) Normal mouse igg, by Merck Group (3 mentions) Normal rabbit igg, by Merck Group (2 mentions) Sybr green master mix, by Promega (2 mentions) Mab8935, by Merck Group (2 mentions) Protein g dynabead, by Thermo Fisher Scientific (2 mentions) Pab28477, by Abnova (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) H6908, by Merck Group (1 mentions) Mycoplasma detection kit, by Vazyme (1 mentions) Ab198202, by Abcam (1 mentions) Ab9100, by Abcam (1 mentions)

5 protocols using ab57154

1

ChIP Assay for Transcription Factor Binding

Cited 2 times
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The binding of transcription factors (TFs) on target promoters was evaluated by performing the ChIP assay, as described previously (29 (link)). Briefly, lysed cells were sonicated (approximate chromatin fragment length: 200–500 bp), and chromatins (25 μg) were immunoprecipitated with concomitant antibody followed by overnight incubation at 4° C. The following antibodies were used for ChIP: anti-human pSMAD2/3 (S465/S467) (R&D Systems, MAB8935, lot no. CJRA0318051), anti-SLUG (Sigma, PRS3959, lot no. 87711801), anti-RBFOX2 (Abcam, ab57154, batch no. GR317972-9), normal rabbit IgG (Millipore, 12-370, lot no. 2295402) and normal mouse IgG (Millipore, 12-371B, lot no. 2332526). The immunoprecipitated protein–DNA complexes and 5% input were analyzed by qRT-PCR in triplicate using specific primers (Supplementary Table S10) flanking the predicted binding sites and SYBR Green Master Mix (Promega, A6002, lot no. 0000385100). All the ChIP experiments were performed at least thrice. IP values were normalized to input using the following formula: 2^(Ct_input – Ct_immunoprecipitation) (30 (link)). Resultant values were subsequently normalized to IgG control IP values. The significance between two different groups was identified using Student’s t-test. P < 0.05 was considered statistically significant.
Ahuja N., Ashok C., Natua S., Pant D., Cherian A., Pandkar M.R., Yadav P., Narayanan S.S. V., Mishra J., Samaiya A, & Shukla S. (2020). Hypoxia-induced TGF-β–RBFOX2–ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer. NAR Cancer, 2(3), zcaa021.
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2

Chromatin Immunoprecipitation Assay for TF Binding

Cited 2 times
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The binding of transcription factors (TFs) on target promoters was evaluated by performing the ChIP assay, as described previously (29 (link)). Briefly, lysed cells were sonicated (approximate chromatin fragment length: 200–500 bp), and chromatins (25 μg) were immunoprecipitated with concomitant antibody followed by overnight incubation at 4°C. The following antibodies were used for ChIP: anti-human pSMAD2/3 (S465/S467) (R&D Systems, MAB8935, lot no. CJRA0318051), anti-SLUG (Sigma, PRS3959, lot no. 87711801), anti-RBFOX2 (Abcam, ab57154, batch no. GR317972-9), normal rabbit IgG (Millipore, 12-370, lot no. 2295402) and normal mouse IgG (Millipore, 12-371B, lot no. 2332526). The immunoprecipitated protein–DNA complexes and 5% input were analyzed by qRT-PCR in triplicate using specific primers (Supplementary Table S10) flanking the predicted binding sites and SYBR Green Master Mix (Promega, A6002, lot no. 0000385100). All the ChIP experiments were performed at least thrice. IP values were normalized to input using the following formula: 2^(Ct_input − Ct_immunoprecipitation) (30 (link)). Resultant values were subsequently normalized to IgG control IP values. The significance between two different groups was identified using Student’s t-test. P < 0.05 was considered statistically significant.
Ahuja N., Ashok C., Natua S., Pant D., Cherian A., Pandkar M.R., Yadav P., Narayanan S.S. V., Mishra J., Samaiya A, & Shukla S. (2020). Hypoxia-induced TGF-β–RBFOX2–ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer. NAR Cancer, 2(3), zcaa021.
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3

SLUG-RBFOX2 Interaction Characterization

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The SLUG and RBFOX2 interactions were evaluated by performing co-immunoprecipitation (co-IP), as described previously (31 (link)). Briefly, the hypoxia-treated MCF7 cells were lysed using a lysis buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40 and protease inhibitor cocktail (PIC; Roche)]. The lysates were incubated with anti-SLUG (Sigma, PRS3959, lot no. 87711801) or anti-RBFOX2 (Abcam, ab57154, batch no. GR317972-9) antibody along with normal rabbit IgG (Millipore, 12-370, lot no. 2295402) or normal mouse IgG (Millipore, 12-371B, lot no. 2332526) for 2 h at 4°C. Subsequently, 25 μl Protein-G Dynabeads (Thermo Fisher Scientific, 10004D, lot 0078227) were added to the immunoprecipitated lysate and further incubated for 8 h at 4°C. The beads were then washed thrice with the lysis buffer and boiled with 2× Laemmli buffer for 5 min. Immunoblotting analysis was performed with the eluted proteins with anti-SLUG and anti-RBFOX2 antibodies.
Ahuja N., Ashok C., Natua S., Pant D., Cherian A., Pandkar M.R., Yadav P., Narayanan S.S. V., Mishra J., Samaiya A, & Shukla S. (2020). Hypoxia-induced TGF-β–RBFOX2–ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer. NAR Cancer, 2(3), zcaa021.
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4

Evaluating SLUG-RBFOX2 Interactions by Co-IP

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The SLUG and RBFOX2 interactions were evaluated by performing co-immunoprecipitation (co-IP), as described previously (31 (link)). Briefly, the hypoxia-treated MCF7 cells were lysed using a lysis buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40 and protease inhibitor cocktail (PIC; Roche)]. The lysates were incubated with anti-SLUG (Sigma, PRS3959, lot no. 87711801) or anti-RBFOX2 (Abcam, ab57154, batch no. GR317972-9) antibody along with normal rabbit IgG (Millipore, 12-370, lot no. 2295402) or normal mouse IgG (Millipore, 12-371B, lot no. 2332526) for 2 h at 4°C. Subsequently, 25 μl Protein-G Dynabeads (Thermo Fisher Scientific, 10004D, lot 0078227) were added to the immunoprecipitated lysate and further incubated for 8 h at 4°C. The beads were then washed thrice with the lysis buffer and boiled with 2× Laemmli buffer for 5 min. Immunoblotting analysis was performed with the eluted proteins with anti-SLUG and anti-RBFOX2 antibodies.
Ahuja N., Ashok C., Natua S., Pant D., Cherian A., Pandkar M.R., Yadav P., Narayanan S.S. V., Mishra J., Samaiya A, & Shukla S. (2020). Hypoxia-induced TGF-β–RBFOX2–ESRP1 axis regulates human MENA alternative splicing and promotes EMT in breast cancer. NAR Cancer, 2(3), zcaa021.
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5

Cell Line Cultivation and Antibody Validation

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Human NPC cell lines (S26 and 5–8F) were kindly gifted by Professor Chaonan Qian at SYSUCC and human embryonic kidney 293T cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. All cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA) and cell cultures were maintained in a 5% CO2 humidified incubator at 37 °C. No evidence of mycoplasma contamination was observed using mycoplasma detection kit (Vazyme, Nanjing, China). Primary antibodies were commercially available, including those for GOLIM4 (PAB28477, Abnova, Taipei, China), β‐actin (A1978, Sigma‐Aldrich, St. Louis, USA) and RBFOX2 (HPA006240, Sigma‐Aldrich, St. Louis, USA, ab57154, Abcam, Cambridge, USA), Tubulin, CCND1, C‐MYC, N‐Cadherin, and Vimentin (2144, 2922, 9402, 14215, 5741, Cell Signaling Technology, Danvers, USA), RAB26 (ab187151, ab198202, Abcam, Cambridge, USA), E4F1 (H00001877‐M03, Abnova, Taipei, China) and HA tag (ab9100, Abcam, Cambridge, USA, H6908, Sigma‐Aldrich, St. Louis, USA).
Luo C., Xu X., Liu C., He S., Chen J., Feng Y., Liu S., Peng W., Zhou Y., Liu Y., Wei P., Li B., Mai H., Xia X, & Bei J. (2021). RBFOX2/GOLIM4 Splicing Axis Activates Vesicular Transport Pathway to Promote Nasopharyngeal Carcinogenesis. Advanced Science, 8(16), 2004852.
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