Protein samples were separated on a 4–15% TGX gel (Bio-Rad, USA) and blotted onto a PVDF membrane. Membranes were blocked in 5% milk powder dissolved in PBS. Primary antibody was incubated with membrane overnight at 4 °C. Following morning, membranes were washed in PBS + T two times for 10-min and incubated with secondary antibody (Agilent, USA) for 60 min at room temperature. After three times of 5-min washes in PBS+T proteins were detected with Super-Signal West Pico Chemiluminescent (Thermo Fisher Scientific, USA) and developed with a ChemiDoc MP instrument (Bio-Rad, USA). Following antibodies were used in this study: Monoclonal Anti-FLAG M2 antibody (F3165, Anti-Sigma-Aldrich, USA) and Anti-RBP1 antibody (ab140509, abcam, UK). Uncropped gel pictures can be found in the Source Data file.
Chemidoc mp instrument
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc MP instrument is a high-performance imaging system designed for a variety of applications in life science research. It captures images of chemiluminescent, fluorescent, and colorimetric samples, such as western blots, gels, and membranes.
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Lab products found in correlation
Image lab software, by Bio-Rad (7 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Clarity max western ecl substrate, by Bio-Rad (3 mentions)
Gapdh hrp, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by PerkinElmer (2 mentions)
Phospho stat1 y701, by Cell Signaling Technology (2 mentions)
Stat1, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Human primary anti notch cleaved 1, by Cell Signaling Technology (2 mentions)
Purified human anti hes1, by Cell Signaling Technology (2 mentions)
Halt protease phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate kit, by Thermo Fisher Scientific (2 mentions)
Image lab program, by Bio-Rad (2 mentions)
Image lab 5, by Bio-Rad (2 mentions)
Candycane glycoprotein ladder, by Thermo Fisher Scientific (2 mentions)
Nupage bis tris acrylamide gel, by Thermo Fisher Scientific (2 mentions)
31 protocols using chemidoc mp instrument
1
Western Blot Analysis of Protein Samples
2
Protein Sample Preparation and Western Blotting
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Samples were first collected by lysing in either RIPA buffer (50 mM Tris-Cl, pH 8.0; 150 mM sodium chloride; 1% (v/v) Triton X-100; 0.5% (w/v) sodium deoxycholate; 0.1% (w/v) sodium dodecyl sulfate (SDS)) or 1× SDS-PAGE sample buffer. In either case, HALT protease inhibitor cocktail and EDTA were included at 1× and 0.5 mM final concentrations, respectively (Thermo# 78438). In vivo samples were collected as done before by scraping intestinal tissue with a clean scalpel and transferring the liberated epithelium to a fresh microcentrifuge tube (Kao et al., 2017 (link); Zheng et al., 2017 (link)). Samples were sonicated as needed to reduce viscosity. Samples were analyzed by SDS-PAGE using standard techniques (Brunelle and Green, 2014 (link)) and transferred to PVDF for blotting. Blots were blocked in 5% (w/v) nonfat dry milk and probed with antibodies listed in the Key Resources Table . Blots were developed using Clarity Max ECL reagent and imaged using a Bio-Rad ChemiDoc MP instrument.
3
Protein Analysis of ACBI1 Treated Cells
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Cells were treated with 250 nM ACBI1, cis-ACBI1, or matched DMSO control for 24 h and then harvested for protein lysate preparation. To make lysates, cold lysis buffer containing 5 mM EDTA, 150 mM NaCl, 150 mM Tris, pH 8.0, 1% Triton X-100, 0.001M PMSF, and 1X Roche protease inhibitor cocktail was used to collect the cells after treatment. The cells were sonicated for 15 s at 25% power and centrifuged to remove cellular debris. To determine protein concentration, the BioRad Bradford assay was used with bovine serum albumin as a standard protein. Then, 15–30 μg of protein per sample was separated by SDS-PAGE and then transferred to a PVDF membrane (PerkinElmer, Shelton, CT, USA). Membranes were placed in 5% milk made in TBS-T (150 mM NaCl, 50 mM Tris, 0.1% Tween-20) to block unspecific interactions. The antibodies used for immunoblotting were BRG1 (Cell Signaling, 49360, Danvers, MA, USA), GAPDH-HRP (Cell Signaling, 8884), JUN (Cell Signaling, 9165), JUNB (Cell Signaling, 3753), and JUND (Cell Signaling, 5000). The Clarity ECL substrate (BioRad, Hercules, CA, USA) was used to visualize bands on a Bio-Rad Chemidoc MP instrument.
4
Protein Sample Preparation and Western Blotting
5
Protein Expression Analysis Protocol
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Equal amount of proteins from cultured cells and clinical tissues were separated by SDS-PAGE and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, USA). The membrane was then probed with a primary antibody including anti-CtBP1 (mouse, sc398945), anti-CtBP2 (goat, sc5967), anti-Bax (mouse, sc20067), anti-Bim (mouse, sc374358), anti-E-cadherin (mouse, sc21791), anti-PUMA (mouse, sc377015), anti-p16 (mouse, sc166760), anti-p21 (mouse, sc6246) or anti-PTEN (mouse, sc7974), followed by probing with a peroxidase-conjugated secondary antibody. All of the primary antibodies were purchased from Santa Cruz Biotechnology. The signals were detected with the ChemiDoc MP instrument (Bio-Rad Laboratories, USA).
6
Immunoblotting of Cell Signaling Proteins
7
Quantitative Western Blot Analysis
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Treated cells were washed with PBS then lysed in RIPA buffer (Cat #R0278, Sigma) with protease (Cat #P8340, Sigma) and phosphatase inhibitors (Cat #P5726, Sigma). Protein content was quantified using the BCA assay. Proteins were electrophoresed on a 4–12% polyacrylamide gel and then transferred to a PVDF membrane, blocked with non-fat dried milk or BSA, and incubated with primary antibody at overnight at 4°C according to manufacturer recommendations. Cell Signaling Technology primary antibodies used in these studies were: STAT1 (Cat #9175), Phospho-STAT1 (Y701) (Cat #7649), STAT3 (Cat #9139), Phospho-STAT3 (Tyr 705) XP (Cat #9145), STAT5 (Cat #9363), Phospho-STAT5 (Tyr694) (Cat #9351), JAK2 XP (Cat #3230), Phospho-JAK2 (Tyr1007) (Cat #4406), and β-actin (Cat #3700). After the membrane was washed, it was incubated with secondary antibody (Cell Signaling Technology, anti-rabbit IgG-HRP linked #7074 or anti-mouse IgG-HRP linked #7076) for 1 h then treated with ECL or Femto substrate. Images were captured with a BioRad ChemiDoc MP instrument and analyzed using Image Lab software (BioRad).
8
Western Blot Quantification Protocol
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For western blot, a standard SDS-PAGE gel was prepared according to the Laemmli protocol20 (link) with modifications according to Ladner et al.21 (link). Precision Plus Protein™ Dual Xtra (Bio-Rad Laboratories) was used as molecular weight standard in 1:10 dilution. The proteins in the gel were transferred to a low-fluorescence PVDF blotting membrane in a semi-dry blot procedure (15 min, 25 V. 1.3 A, Trans-Blot® TurboTM instrument, Bio-Rad Laboratories) using the Trans-Blot® TurboTM RTA transfer kit. The membrane was blocked with AdvanBlock™ -PF blocking solution for 30 min, incubated with “8-4-4” anti-His-tag antibody (3.3 mg/L in blocking solution, 60 min) and washed with AdvanWashTM washing solution (3 × 5 min). Then, the membrane was incubated with secondary RPE-goat-anti-mouse-IgG (1:4000 in washing buffer with 0.5% milk powder, 60 min) and washed with washing solution (2 × 5 min) and finally with Tris buffer (20 mmol/L Tris, 500 mmol/L NaCl, pH 7.5, 1 × 5 min). Fluorescence detection was performed using the Chemi-Doc MP instrument (Bio-Rad Laboratories) by measuring the Cy3 channel (605/50 nm, recording time 5.33 s).
9
Western Blot Protein Expression Analysis
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The expression levels for each protein were monitored by western blot. For western blot, the gel was obtained through the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) process. The proteins in the gel were transferred to a low-fluorescence PVDF blotting membrane in a wet blotting procedure (2 h, 100 V, 400 mA). The blotting was then blocked with 5% milk in TBST for 1 h. The blotting was then incubated with the primary anti-His-tag antibody (1:2000 in blocking solution) and washed with 1 × TBST washing solution (3 × 5 min). Then, the membrane was incubated with secondary RPE-goat-anti-mouse-IgG (1:5000 in washing buffer, 60 min) and washed with washing solution (3 × 5 min). Fluorescence detection was performed using the Chemi-Doc MP instrument (Bio-Rad Laboratories). Blots were imaged using an Azure Biosystems c280 imager.
10
Quantitative Protein Expression Analysis
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The whole-cell protein was extracted by using ice-cold RIPA buffer mixed with protease inhibitors. The protein lysate was separated by SDS-PAGE at 110 V for 100 min and transferred to PVDF membranes at 90 V for 90 min. The PVDF membrane was blocked in 5% non-fat milk for 1 h at room temperature, and antibody (MBTD1, ab170848, Abcam, USA, 71 kD; GAPDH, ab9484, 36 kD) was added at 4°C overnight. The membrane was further probed with the secondary antibody IgG H&L (HRP) (ab6721, Abcam, San Francisco, USA, 1:5000) after washing the membrane with PBST (PBS with 0.2% Tween 20). The protein bands were detected by Pierce ECL Plus western blotting substrate (Thermo Fisher, Waltham, USA) on a ChemiDoc MP instrument (Bio-Rad, California, USA).
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