Cell lysates and supernatants were loaded on 12% criterion TGX gels according to manufacturer’s instructions (Bio-Rad). Following SDS-PAGE, samples were transferred onto a 0.22 µm nitrocellulose membrane using the criterion blotting system (Bio-Rad). All washing and incubation steps have been described previously37 (link). For detection of O-tagged VNAs, the following antibodies have been used: OLLAS Epitope Tag Antibody (L2) from rat (Novusbio, 1:1,000) and a Rabbit anti-α/β tubulin (New England Biolabs, 1:1000) as primary antibodies, and Goat anti-Rat IgG (H + L) IRDye 800 CW (LI-COR, 1:15,000) as well as a Goat anti-Rabbit IgG (H + L) IRDye 680 RD (LI-COR, 1:15,000) as secondary antibodies. Primary antibodies were incubated for two hours. Secondary antibodies were incubated for one hour. For detection of RNA-encoded EfAb the following antibodies have been used: A Rabbit anti-tubulin UNLB (Cell Signaling Technology, 1:15,000) as primary antibody, a Goat anti-human IgG CW800 (LI-COR, 1:15,000) as well as a Goat anti-rabbit RD680 (LI-COR, 1:15,000) as secondary antibodies. Primary and secondary antibodies have been incubated for one hour. Protein detection and image processing were carried out in an Odyssey CLx® Imaging system and Image Studio version 5.2.5 (LI-COR) according to manufacturer’s recommendations.
Image studio version 5
Manufactured by LI COR
Sourced in Germany, United States
Image Studio version 5.2 is a software application designed for the analysis and quantification of gel and blot images. It provides tools for image acquisition, processing, and analysis to support a wide range of life science research applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx imaging system, by LI COR (7 mentions)
Odyssey clx infrared imaging system, by LI COR (5 mentions)
Odyssey clx, by LI COR (4 mentions)
Odyssey fc, by LI COR (3 mentions)
Semi dry blotter, by Bio-Rad (3 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Prism 7, by GraphPad (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Secondary antibodies with, by LI COR (2 mentions)
Conjugated 680nm and 800nm fluorophores, by LI COR (2 mentions)
Kdm4b jmjd2b, by Fortis Life Sciences (2 mentions)
Top2a, by Santa Cruz Biotechnology (2 mentions)
Top2b, by Santa Cruz Biotechnology (2 mentions)
Mdr1 abcb1, by Cell Signaling Technology (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Typhoon imager, by GE Healthcare (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
Odyssey clx system, by LI COR (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
74 protocols using image studio version 5
1
Western Blot Analysis of O-Tagged VNAs and RNA-Encoded EfAb
2
Western Blot Profiling of Nuclear Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear or Whole Cell Extracts were prepared, and protein concentrations were measured. 15ug of protein was run on a Bis-Tris gel, transferred at 200mA for 2 hours and blocked in 5% non-fat milk for 1 hour. Blots were incubated with primary antibodies overnight and then washed 3x in TBST. Blots were then incubated with secondary antibodies with conjugated −680nm and −800nm fluorophores (Licor, 1:25,000) for 45 minutes at room temperature, washed 3x, and then developed on an Odyssey® CLx Imaging System (Licor, Lincoln, NE United States). KDM4B/JMJD2B (Bethyl Laboratories A301–478A, 1:1000), TOP2A (Santa Cruz Biotech sc-3659, clone F-12, 1:1000), TOP2B (Santa Cruz Biotech H-286, sc-13059, 1:1000), MDR1/ABCB1 (Cell Signalling Technologies, 12683S, 1:1000), RNF2 (Cell Signalling Technologies, 5694S, 1:1000), H3 (Epicypher, SKU: 13–0001, 1:5000) We were unable to validate any of the available KAT6B antibodies. Licor software (Image Studio version 5.2.5) was used for the following: Image adjustment was limited to exposure (brightness and contrast; all lanes done equally and simultaneously) followed by cropping and exporting in .png format. No additional image manipulation was performed.
3
Comprehensive Immunological Data Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Data were analyzed using GraphPad Prism software version 6. All error bars depicted in this publication refer to standard deviations. ELISA data were analyzed using nonlinear regression. FACS plots were interpreted by BD FACSDiva™ software version 6.1.3 (Version), and data were further processed by FlowJo™ software version 10.0.7. Cells were gated on all living cells, and median PE fluorescence was calculated. Whole image processing was carried out using LI‐COR's Image Studio version 5.2.5 according to manufacturer's recommendations. For correlation analysis of IgG titers and mouse anti‐SO57 antibodies, a two‐tailed nonparametric spearman correlation with a confidence interval of 95% was used.
4
DNA Quantification and Immunoblot Analysis
5
Quantitative Immunoblot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction with trichloroacetic acid, immunoblotting, and protein detection by immunoblotting using a Li-Cor Odyssey infrared dye detection system were performed as previously described (20 (link)). Protein abundance was quantified from immunoblots using Image Studio version 5.2.5 (LiCor) (licor.com/bio/image-studio/ ). Rabbit anti-Pyk1 antibody (69 (link)) was a gift of Jeremy Thorner, and rabbit anti-Fba1 antibody (70 (link)) a gift of Magdalena Boguta. Anti-Pgk1 (22C5D8) was obtained from Abcam. IRDyeR 680LT dye-labeled anti-mouse antibody and IRDyeR 800CW dye-labeled anti-rabbit antibody were obtained from Li-Cor. Antibody specificity in immunoblots was verified by comparison of GFP-tagged and untagged target molecular weight via immunoblotting, and/or by comparison of protein samples from WT and deletion mutant strains.
6
Western Blot Profiling of Nuclear Factors
7
Western Blot Analysis Methodology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For all experiments, pooled triplicates of equal amounts of lysates or supernatants were loaded. For all western blots depicted in this study, 12% SDS Tris-glycine gels were used. Proteins were transferred to a nitrocellulose membrane (Odyssey nitrocellulose membrane, 0.22 μm; Li-COR Biosciences, cat. no. 926-31092) and afterward blocked in 5% skimmed milk in TBST buffer (TBS containing 0.1% Tween 20; Sigma, cat. no. P2287). Membranes were first incubated with rabbit anti-a/b-tubulin 1:1,000 (New England Biolabs, cat. no. 2148S) in 0.5% skimmed milk in TBST for 1 h. After three washes (10 min each) in TBST, both a secondary antibody against rabbit (goat anti-rabbit IgG [H + L] IRDye 680RD; Li-COR Biosciences, cat. no. 926-68071) and an antibody to detect human antibodies (goat anti-human IgG [H + L] IRDye 800CW; Li-COR Biosciences, cat. no. 926-32232) were incubated at 1:15,000 in 0.5% skimmed milk in TBST for 1 h or, in case of HepG2 supernatants, overnight. Immediately before band detection, all membranes were washed three times each for 10 min in TBST and stored in TBS lacking Tween 20 until analysis. Protein detection and image processing were carried out in an Odyssey CLx imaging system and LI-COR’s Image Studio version 5.2.5 according to the manufacturer’s recommendations.
8
Western Blot Analysis of NF-κB Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Experiments were performed according to standard procedures and as previously described.34 (link) The following antibodies were used: anti-P65 (L8F6) mouse monoclonal antibody, 6956, dilution 1:1000; anti-NFKB Inhibitor Alpha (L35A5) mouse monoclonal antibody, 4814 at 1:1000 (Cell Signaling Technology, Danvers, MA); and anti–β-actin rabbit monoclonal antibody, 926-42210 at 1:1000 (LI-COR Biosciences, Lincoln, NE). Western blot images were acquired using a LI-COR Odyssey scanner. Protein band densitometry analysis was performed using LI-COR Image Studio version 5.2 software.
9
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flash-frozen tumors were homogenized in PBS with Halt phosphatase inhibitor (Thermo Fisher Scientific) and protease inhibitor (Sigma-Aldrich) using the Precellys Evolution Homogenizer with Cryolys (Bertin Instruments). The preset elastic setting was used for homogenizing. Tumor homogenates were centrifuged to remove PBS and resuspended in RIPA buffer with phosphatase and protease inhibitors. Lysates were sonicated 2 × 10 s using a Branson digital sonifier at 10% amplitude. Samples were centrifuged at 15,000 rpm for 5 min at 4°C and supernatants collected and quantified by Bradford assay (Bio-Rad). Samples (35 μg total) were blotted for BRAF (sc-5284; 1:500; Santa Cruz) and β-Actin (#3700; 1:1,000; Cell Signaling) and imaged using a LI-COR Odyssey CLx system. Bands were quantified using Image Studio Version 5.2 software (LI-COR Biosciences).
10
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in 1% SDS containing 2% β-mercaptoethanol and boiled for 20 min prior to separation by SDS-PAGE and transfer to Optitran membranes (GE Healthcare). Membranes were blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) followed by incubations in primary and secondary antibody (10 (link)). Membranes were washed in TBST and imaged with the Odyssey Fc imager (Li-Cor). Images were generated with Image Studio version 5.2 software (Li-Cor).
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!