HEK293T cells transfected with different combinations of plasmids were harvested and resuspended in 1 ml ice-cold Pierce IP buffer (87787) containing protease inhibitors (Complete Mini EDTA-free, Roche and PMSF) and phosphatase inhibitor (NaF and NaVO3). Cells were lysed for 30 min at 4 °C and lysates were spun for 30 min at maximum speed at 4 °C. 20–40 μl anti-Flag M2 beads (Sigma, M8823) were added to the cell lysate and incubated overnight. Beads were washed five times with immunoprecipitation buffer and, finally, bound proteins eluted by boiling in loading buffer. Samples were separated on 4–12% Tris-Bis gels (Life Technology) and transferred onto PVDF membranes (Whatman). Western blot analysis was performed using antibodies against Flag (Sigma, F1804 or F7425), HA (Sigma, H3663 or Abcam, ab9110), Casp1 p10 (Santa Cruz, sc-514), DHX9 (Abcam, ab26271), DHX15 (Abcam, ab70454 or ab13311), Casp3 p17/p19 (Cell Signaling, 9661), LC3 (Cell Signaling, 2775), or β-actin (Sigma, A1978).
Ab9110
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Ab9110 is a laboratory instrument designed for protein detection and analysis. It utilizes immunoassay technology to quantify the presence and concentration of specific proteins in biological samples. The core function of Ab9110 is to provide accurate and reliable protein measurement data to support research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 bead, by Merck Group (2 mentions)
Ab26271, by Abcam (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
H3663, by Abcam (1 mentions)
Sc 514, by Abcam (1 mentions)
F1804, by Merck Group (1 mentions)
F7425, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Complete mini, by Roche (1 mentions)
Ez chip kit, by Merck Group (1 mentions)
Avcx130 system, by Sonics (1 mentions)
Anti oct1, by Santa Cruz Biotechnology (1 mentions)
Normal mouse anti igg, by Merck Group (1 mentions)
Anti ha, by Abcam (1 mentions)
Anti h3k4me3, by Merck Group (1 mentions)
Anti rbbp5, by Fortis Life Sciences (1 mentions)
Anti his, by Santa Cruz Biotechnology (1 mentions)
Anti ash2l, by Fortis Life Sciences (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
5 protocols using ab9110
1
HEK293T Cell Immunoprecipitation and Western Blot
2
ChIP-PCR for HA-tagged CP2
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As specifical immunoprecipitate CP2 antibodies are not available, we constructed pCMV-C-HA-CP2-CDS and then transfected this vector into mGCs. ChIP was performed using the EZ-ChIP Kit (Millipore). The AVCX130 system (Sonics & Materials, Newtown, CT, USA) was used for cell sonication. Anti-HA (Abcam, ab9110), anti-OCT1 (Santa Cruz Biotechnology, sc-25399 X) and normal anti-mouse-IgG (Millipore) were used for the immunoprecipitation reactions. DNA from the immunoprecipitated complex was amplified via PCR. The primer sequences are described in Supplementary Table S2 .
3
Antibody Validation for Protein Interactions
4
ChIP Assay and Quantitative PCR Protocol
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Antibodies were described before [27 (link)] or are as follows: anti-HA [12CA5] (Roche, 11583816001, for western); anti-HA (Abcam, ab9110, for ChIP); anti-Myc (Santa Cruz Biotechnology, sc-764x) anti-Oct4 (Santa Cruz Biotechnology, sc-8628x). RNA extraction and ChIP assays were done as described [27 (link)]. qPCR was performed with SYBR Advantage qPCR Premix (Clontech) on a ViiA7 Real-Time PCR System (Applied Biosystems). Primers used are listed in Table 1 .
5
Investigating SIX3 Protein Regulation via Co-Immunoprecipitation
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The details of these procedures were described previously [37 (link)]. Anti-SIX3 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-81985), anti-FLAG (Abcam, Cambridge, MA, USA; ab205606), anti-HA (Abcam; ab9110), and anti-IgG (Santa Cruz Biotechnology; sc-2027) antibodies were used for co-immunoprecipitation (Co-IP). Immunoprecipitates were washed at least five times and subjected to western blot analysis using anti-TRIM27 (Abcam; ab78393), anti-NEDD4 (Abcam; ab236512), anti-SMURF2 (Abcam; ab94483), anti-RNF6 (Abcam; ab204506), anti-SYVN1 (Abcam; ab170901), anti-MDM2 (Abcam; ab16895), and Anti-SIX3 (Abcam; ab172131) antibodies. For ubiquitination assays, the lysates of A549 cells transfected with siTRIM27-1 or siNC were used for IP with an anti-IgG (Santa Cruz Biotechnology; sc-2027) or Anti-SIX3 antibody (Santa Cruz Biotechnology; sc-81985) and Protein A/G PLUS-Agarose (Novex, Oslo, Norway), which was performed at 4° C overnight. The eluted proteins were then detected by western blot analysis using an anti-ubiquitin (Ub) antibody (Abcam, ab7780).
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