GFP (Roche, 11814460001; 1:2000), PAR 10H (Trevigen, 4335-AMC-050; 1:200), PAR binder (Millipore, MABE1031, 1:1000), CTIF (Sigma-Aldrich, HPA016865-100UL; 1:1000), TERF1 (Abcam, ab10579; 1:1000), GLUT4 (Abcam, ab654; 1:1000), ILF3 (Abcam, ab92355; 1:1000), NPM1 (Abcam, ab10530; 1:1000), CETN3 (Abnova, H00001070-M01; 1:500), PCNT (Atlas antibodies, HPA016820; 1:500), PCM1 (Cambridge bioscience, A301-149A; 1:500), gamma-tubulin (Sigma-Aldrich, T6557-100UL; 1:500), Azi1/Cep131 (Abcam, ab84864; 1:500), Cep290 (Abcam, ab84870; 1:500), BBS4 (Proteintech, 12766-1-AP; 1:500), OFD1 (Proteintech, 22851-1-AP; 1:500), TNKS1/2 (Santa Cruz, sc-8337; 1:1000), alpha-tubulin (Sigma-Aldrich, T9026; 1:10000), beta-actin (Sigma-Aldrich, A1978; 1:10000).
Ab92355
Manufactured by Abcam
Ab92355 is a monoclonal antibody that recognizes the protein GAPDH. It is suitable for use in Western Blotting and Immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab10579, by Abcam (1 mentions)
Ab654, by Abcam (1 mentions)
A1978, by Merck Group (1 mentions)
Gamma tubulin, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Alpha tubulin, by Merck Group (1 mentions)
Ab154169, by Abcam (1 mentions)
Ab5821, by Abcam (1 mentions)
Ab45427, by Abcam (1 mentions)
Ab10739, by Abcam (1 mentions)
Ab76818, by Abcam (1 mentions)
Iblot2 system, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ab133607, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Upright microscope, by Leica (1 mentions)
Df3645, by Abcam (1 mentions)
6 protocols using ab92355
1
Immunofluorescence Protein Detection Protocol
2
Western Blot Analysis of Cellular Proteins
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Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 150 mM NaCl), supplemented with protease inhibitor cocktail (Roche), 5 mM NaF and 0.2 mM Sodium orthovanadate. Protein lysates were mixed with reducing agent and LDS sample buffer (Novex, ThermoFisher) and denatured at 70°C for 10 min and loaded in Novex Nupage 4–12% Bis-Tris gels. Gels were transferred onto nitrocellulose membrane using the iBlot2 system (ThermoFisher). Membranes were blocked with PBS-0.05% Tween and 5% milk for 1 h at room temperature with agitation, before overnight incubation with primary antibodies. Antibodies against PKR (ab45427 Abcam), ILF3 (ab92355 Abcam), s6RP (2317S CST), eIF6 (3833S CST), ILF2 (ab154169 Abcam), α-tubulin (CP06 Merck), fibrillarin (ab5821 Abcam), phospho-eIF2α (Ser-51) (D9G8) (3398S CST), IFNAR1 (ab10739 Abcam), IFIT3 (ab76818 Abcam), OASL (ab191701), IRF1 (CST #8478), eIF3M (Bethyl, A305–029A), anti-rabbit HRP (CST) and anti-mouse HRP (Bio-Rad) were used. Proteins were visualized using ECL (Pierce) on a Bio-Rad ChemiDoc imaging system. Protein bands were quantified using ImageJ (v1.51p) software and normalized to α-tubulin or fibrillarin.
3
RNA-Protein Binding Assay by EMSA
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For EMSA assays, synthesized RNA labelled with a 5′fluorescent DY781 dye and containing a three base extension was used (5′-DY781-AGUGGCUUAUGCCUGUA/GAUCCCAACAC). Unlabeled RNA was used for competition assays.
Protein-RNA-binding reactions, containing 1 μM labelled RNA and 3 μM ILF3 or hnRNPC in a 10 μl reaction volume, were carried out on ice for 45 min. Competition assays were also carried out in a 10-μl reaction volume. A 1:1 molar ratio complex of RNA and proteins was prepared by incubation on ice for 1 h. The complex at a final concentration of 0.5 μM was mixed with 50 μM unlabeled competitor RNA. Monoclonal antibodies specific for ILF3 (Abcam, ab92355) and hnRNPC (Abcam, ab133607) were added to the binding reaction after complex formation. A 6% polyacrylamide gel in 0.5× Tris/Borate/EDTA buffer (TBE) was pre-run at 4°C for 1 h. Samples were then mixed with 2 μl of native gel loading buffer to a total volume of 6 μl and run on the gel for 2 h. The gel was scanned on a LICOR Odyssey fluorescent infrared scanner at 800 nm. Images were converted to greyscale by ImageJ.
Protein-RNA-binding reactions, containing 1 μM labelled RNA and 3 μM ILF3 or hnRNPC in a 10 μl reaction volume, were carried out on ice for 45 min. Competition assays were also carried out in a 10-μl reaction volume. A 1:1 molar ratio complex of RNA and proteins was prepared by incubation on ice for 1 h. The complex at a final concentration of 0.5 μM was mixed with 50 μM unlabeled competitor RNA. Monoclonal antibodies specific for ILF3 (Abcam, ab92355) and hnRNPC (Abcam, ab133607) were added to the binding reaction after complex formation. A 6% polyacrylamide gel in 0.5× Tris/Borate/EDTA buffer (TBE) was pre-run at 4°C for 1 h. Samples were then mixed with 2 μl of native gel loading buffer to a total volume of 6 μl and run on the gel for 2 h. The gel was scanned on a LICOR Odyssey fluorescent infrared scanner at 800 nm. Images were converted to greyscale by ImageJ.
4
Quantitative Immunoblotting of ILF3 Isoforms
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Mouse tissue extracts were prepared by homogenization and sonication in Laemmli sample buffer. After boiling for 5 min, the extracts were loaded onto SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany). The membranes were probed with anti-ILF3 rabbit monoclonal antibody (ab92355, Abcam, Cambridge, UK), anti-ILF3 rabbit polyclonal antibody (19887-1-AP, Proteintech, Chicago, IL), and anti-α-tubulin rabbit polyclonal antibody (PM054, Medical & Biological Laboratories, Nagoya, Japan). Biotinylated secondary antibody (Cytiva, Marlborough, MA) and alkaline phosphatase-conjugated streptavidin (Cytiva) were used for the detection of protein bands in a bromochloroindolyl phosphate/nitro blue tetrazolium solution. Quantitative comparisons of band intensities between the genotypes were performed as previously described using standard curves generated from the dilution series of each tissue extract from the Ilf3+/+ mice on the same membrane.49 (link) The NFAR2/NFAR1 band intensity ratio was calculated by measuring the NFAR1 and NFAR2 bands in the same lane using ImageJ software.71 (link)
5
Immunohistochemical analysis of PSMD3, ILF3, and Ki67
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The overnight incubation of tissue sections from clinical patients or xenografts was made with anti-PSMD3 antibody (Affinity biosciences, DF3645, 1:100), anti-ILF3 (Abcam, ab92355, 1:100), and anti-Ki67 at 4 °C. 0.3% H2O2 was used for blocking endogenous peroxidases for 15 min. After three times of section washing, streptavidin and biotin-conjugated HRP was put into the tissues for incubation for 1 h at 25℃ and then stained with 3’-diaminobenzidine for 30 min and stopped by 5-minute rinsing in H2O. IHC staining results were imaged with a Leica upright microscope.
Two experienced pathologists conducted an independent assessment of immunohistochemical outcomes, which were then scored based on Friedrich’s standard. The ratio of positive tumor cells was assessed with a mark of 0 (0–5%), 1 (6–20%), 2 (21–50%), or 3 (51–100%). The marks of the staining intensity and the ratio of positive cells were multiplied to obtain the final score. A final score ≤ 1 indicated low expression, while a score > 1 was defined as a high expression.
Two experienced pathologists conducted an independent assessment of immunohistochemical outcomes, which were then scored based on Friedrich’s standard. The ratio of positive tumor cells was assessed with a mark of 0 (0–5%), 1 (6–20%), 2 (21–50%), or 3 (51–100%). The marks of the staining intensity and the ratio of positive cells were multiplied to obtain the final score. A final score ≤ 1 indicated low expression, while a score > 1 was defined as a high expression.
6
Tissue Microarray and IHC Analysis of ILF3
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Surgical tissue samples was fixed with 10% formalin for 24 h at room temperature. Formalin-fixed paraffin-embedded surgical tissue samples were used for TMAs and IHC. To construct the TMAs, tissue cylinders (2 mm in diameter) were removed from each sample and selected tissue cylinders were grouped into a single array block using an Unitma Quick-Ray tissue microarrayer (cat. no. UT06; Unitma Co., Ltd). Each TMA specimen was subsequently cut into 4 µm tissue sections, which were mounted on microscope slides. IHC staining was performed as previously described (21 (link)), except an anti-ILF3 antibody (ab92355; 1:400; Abcam) was used as the primary antibody. A total of two pathologists blindly evaluated the percentage of ILF3-positive samples using the NDP.view2 software (version 2.6.13; Japan SLC, Inc.), as well as the intensity of ILF3 IHC staining. A semi-quantitative immunoreactivity scoring system was used to evaluate the staining (22 (link)). The semi-quantitative H-score (0–300) was calculated as the product of the intensity (0, negative; 1, weak; 2, moderate; 3, strong) and the percentage of ILF-3-positive samples (0–100).
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