Tumor pieces were homogenized in 1 mL RIPA buffer [25 mM Tris-Cl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 1x cOmplete protease inhibitor (Roche, Basel, Switzerland)] using a GentleMACS tissue homogenizer (Miltenyi Biotec, Bergisch Gladbach, Germany). For tissue culture cells, cells were scraped in 300 μL RIPA buffer. In each case, the resulting lysate was clarified by centrifugation at 21000 × g for 20 min. Protein concentration of the lysate was determined by BCA assay (Thermofisher). Lysates were resuspended at 1 mg/mL in Laemmli SDS-PAGE sample loading buffer (10% glycerol, 2% SDS, 60 mM Tris-Cl pH 6.8, 1% b-mercaptoethanol, 0.01% bromophenol blue) and denatured at 100°C for 5 min. Extracts (30 μg of protein) were resolved by SDS-PAGE using 12% acrylamide gels running at 120 V until the dye front left the gel. After SDS-PAGE resolution, protein extracts were transferred to nitrocellulose using an iBlot semi-dry transfer system (Thermofisher). Membranes were blocked in 5% non-fat dry milk, incubated in primary antibodies to PHGDH (Sigma-Aldrich, St. Louis, MO, HPA021241, 1:1000) or vinculin (Abcam, Cambridge, MA, ab18058, 1:250) and detected using HRP-conjugated secondary antibodies and chemiluminescence.
Iblot semi dry transfer system
Manufactured by Thermo Fisher Scientific
The IBlot semi-dry transfer system is a compact and efficient device used to transfer proteins from polyacrylamide gels to membrane supports, such as nitrocellulose or PVDF. It utilizes a semi-dry blotting method to facilitate the transfer process, allowing for rapid and consistent protein transfer.
Automatically generated - may contain errors
Lab products found in correlation
Bradford protein assay, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Ab18058, by Abcam (1 mentions)
Gentlemacs tissue homogenizer, by Miltenyi Biotec (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Hpa021241, by Abcam (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Mouse anti human β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Irdye 800cw, by Abcam (1 mentions)
Irdye 680rd, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Runx2, by Abcam (1 mentions)
Odyssey clx digital imager, by LI COR (1 mentions)
Runx1, by Abcam (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Fibronectin, by BD (1 mentions)
E cadherin, by BD (1 mentions)
α β tubulin, by Cell Signaling Technology (1 mentions)
8 protocols using iblot semi dry transfer system
1
Protein Extraction and Western Blot
2
Pull-down Assay for SNX27-VPS26A Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GST-tagged SNX27 PDZ domain (1 nmol) was mixed with VPS26A proteins (1 nmol) in 500 µl 25 mM Tris, pH 8, 300 mM NaCl, and 1 mM DTT and bound to 25 µl glutathione Sepharose resin. After 2-h incubation at 4°C, the resin was washed four times with pull-down buffer and bound proteins eluted in SDS-PAGE sample buffer. Western blot analysis was done using a nitrocellulose membrane and the iBlot semi-dry transfer system (Thermo Fisher Scientific). His-tagged proteins were detected by ECL on photographic film, using a primary mouse anti-penta-His antibody (34660; QIAGEN) and goat anti-mouse HRP-coupled secondary antibody (A16072; Thermo Fisher Scientific).
3
Western Blot Analysis of FOXO1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested, washed twice with cold PBS, lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific), and incubated on ice for 30 min with vortexing for 10 s at 15-min intervals. The protein concentration was determined using a Bradford protein assay (Bio-Rad Laboratories), after which equal amounts of total protein were run on poly-acrylamide gels that were prepared in-house. After electrophoresis, the proteins were transferred using an iBlot semi-dry transfer system (Thermo Fisher Scientific). The following primary antibodies was used: mouse anti-human FOXO1 (1452T, Cell Signaling), rabbit anti-human pFOXO1Ser-329 (PA5-38275, Invitrogen) and mouse anti-human β-actin (sc-47778, Santa Cruz Biotechnology, Inc.). The following secondary antibodies were used: goat anti-rabbit IgG H&L (IRDye® 800CW, ab216773, Abcam) and goat anti-mouse IgG H&L (IRDye® 680RD, ab216776, Abcam). The IRDye® infrared fluorescent dye signal on the cellulose membranes was detected and calculated using an Odyssey® CLX imaging system and Image Studio software, respectively.
4
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blot, lysates were collected on-plate with 1× radioimmunoprecipitation assay buffer (EMD Millipore, #20-188) with protease and phosphatase inhibitors (Thermo Fisher Scientific, #1861280). Lysates were sonicated and cleared before quantification with a Bradford Protein assay (Bio-Rad) and loaded at 50 μg per lane and run on a NuPage bis-tris gel (Thermo Fisher Scientific) and transferred to nitrocellulose membrane with the iBlot semidry transfer system (Thermo Fisher Scientific) and blocked in 5% milk in Tris Buffered Saline + Tween 20 (TBST) before staining with fibronectin (BD Biosciences, #610078; 1:10,000), ZEB1 (LSBio, #LS-C288694; 1:2000), E-cadherin (BD Biosciences, #610182; 1:1000), vimentin [Cell Signaling Technology (CST), #5741; 1:2000], RUNX1 (CST, #4336; 1:2000), RUNX2 (CST, #12556; 1:2000), RUNX3 (CST, #9647; 1:2000), CBFb (Abcam, ab33516; 1:2000), Snail (CST, #3879; 1:1000), Twist1/2 (Abcam, ab50887: 1:50), and CoxIV (CST, #11967; 1:2000) overnight in 5% milk in TBST. LI-COR secondary antibodies, IRDye goat anti-rabbit and goat anti-mouse, 800CW (LI-COR, #925-32219), and 680RD (LI-COR, #925-68076), were applied at 1:10,000 for 1 hour at room temperature in 5% milk in TBST before imaging on the LI-COR Odyssey CLx Digital Imager.
5
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots were performed as previously described (Chung et al., 2016 (link)). Briefly, proteins were extracted from cell pallets with RIPA buffer (Sigma, cat# R0278) containing protease inhibitor (Roche, cat# 04693159001). The concentrations of extracted cellular proteins were measured using a QuantiPro BCA assay kit (Sigma-Aldrich, cat# QPBCA). Equal amounts of protein were loaded into NuPage Bis-Tris gels (Life Technologies, cat# NP3023BOX) and transferred to a polyvinylidene difluoride membrane with iBlot semi-dry transfer system (Invitrogen, cat# IB21001). The membranes were blocked with 5% BSA in TBST and primary antibody was incubated overnight at 4°C. After incubation with secondary antibodies conjugated with horseradish peroxidase, protein bands were visualized using the G:Box BioImaging systems and quantified using the GeneTools image scanning and analysis package. Protein expression was normalized to α/β-tubulin (rabbit polyclonal, 1:2000; Cell Signaling #2148), which serves as a loading control.
6
Western Blot Analysis of Drug Transporters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thirty μg of protein lysate from each cell line was separated by SDS gel electrophoresis (Invitrogen). Proteins were transferred to a nitrocellulose membrane using the iBlot semi-dry transfer system (Invitrogen). Blots were incubated overnight in 5% milk/PBS-Tween containing P-gp and MDR3 primary antibody C219 ALX-801-002 (ENZO Life Sciences) (1:1000) or BCRP primary antibody ALX-BXP-21 (ENZO Life Sciences). C219 recognises amino acid sequences (VQEALD and VQAALD) found in P-gp (170 kDA) and MDR3 (140 kDa). ALX-BXP-21 does not cross react with P-gp, MRP-1 or MRP2 according to manufacturer. Primary antibodies were detected using peroxidase-conjugated anti-mouse IgG secondary (1:2000, Sigma). Chemiluminescence was visualized through exposure to Luminol (Santa Cruz) and X-ray film (Sigma).
7
Immunoprecipitation of TRIM25-NS1 Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were transfected with 3FLAG-2CARD, TRIM25, and NS1 in a 6-well plate. Twenty-four to 48 h post-transfection, cells were washed once in cold PBS and lysed in plates using an NP-40-based lysis buffer [0.5% NP-40, 150 mM NaCl, 50 mM Tris pH 7.5, 5 mM MgCl2, protease inhibitors (EDTA-free mini tablets, Pierce)] for 45 min at 4 °C. Cell lysates were cleared by centrifugation (14,500 × g, 15 min, 4 °C). Lysates were subjected to FLAG immunoprecipitation using FLAG M2 agarose beads (Sigma Aldrich) for 2 h at 4 °C. Immunoprecipitates were washed three times in lysis buffer and resolved on 4–12% NuPAGE Bis-Tris precast gels (Invitrogen), transferred to PVDF membrane using the iBlot semi-dry transfer system (Invitrogen), blocked in either 1% non-fat dried milk or 2% BSA (Anti-Ubiquitin) in TBS-T (TBS, 0.1% Tween-20), and incubated with the relevant primary antibodies: Anti-FLAG HRP (A8592, Sigma Aldrich, 1:10,000 dilution), Anti-TRIM25 HRP (ab200788, AbCam, 1:5000 dilution), Anti-NS1 HRP (NS1-23–1) (sc130568, Santa-Cruz Biotechnology, 1:500 dilution), Anti-Ubiquitin HRP (PD41) (sc8017, Santa-Cruz Biotechnology, 1:500 dilution). Uncropped scans of western blots are shown in Supplementary Fig. 9 .
8
Quantifying Inflammatory Cytokines in Colonic Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Colonic tissue samples were added per well and separated by NuPAGE 4-12% Bis-tris Gel (Novex) for 2 hr at 80 V and then blotted on polyvinlydene fluoride (PVDF) membrane (Novex, 2 Transfer Stack, PVDF, Mini) by the iBlot semi-dry transfer system (Invitrogen). Membranes were blocked in 5% non-fat milk in phosphate buffered saline containing 0.01% Tween 20 (TBST) for one hour and then incubated overnight at +4 °C with anti-beta actin (Bioss, bs-0061R, 1:100), anti-IL-17 (Abcam, ab79056, 1:100) and anti- IL-22 (Bioss, bs-2623R, 1:100). Protein bands were detected using an enhanced chemilu-minescence kit (WesternBreeze kit, Life Techno-logies), quantified by ChemiDoc MP System with the Image Lab software (Bio-Rad) and expressed as the relative intensity of target protein to that B-actin control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!