1×105 MSCs were lysed in non-denaturing lysis buffer (20 mM Tris HCl pH8, 137 mM NaCl, 10% glycerol, 1% TritonX100, 2 mM EDTA) supplemented with protease inhibitor cocktail (Roche). Samples and Protein G sepharose beads (Sigma) were blocked 1h on ice with PBS 1% BSA. Samples were incubated with anti-MIF (1:100, Santa Cruz) overnight at 4°C. Protein G sepharose beads were added to the sample-antibody mix and incubated for 6 h at 4°C. After extensive washes, the pellet was resuspended in SDS loading buffer, boiled and supernatant recovered. These were then processed for western blot analysis and analyzed for the presence of MIF, CXCR2, CXCR4 and CD74.
Anti mif
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-MIF is a laboratory reagent used in research applications. It functions as an antibody that specifically binds to the MIF (Macrophage Migration Inhibitory Factor) protein. The core purpose of Anti-MIF is to facilitate the detection, quantification, or isolation of MIF in various experimental and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd74, by Santa Cruz Biotechnology (2 mentions)
Anti ddt, by Abcam (2 mentions)
Protein g sepharose beads, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Image acquisition system, by Bio-Rad (1 mentions)
Anti f actin, by Abcam (1 mentions)
Epr3776, by Abcam (1 mentions)
Anti p cofilin, by Cell Signaling Technology (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Anti tnf α, by Merck Group (1 mentions)
Mouse anti rhodopsin, by Merck Group (1 mentions)
Mouse anti il 1β, by Merck Group (1 mentions)
Rabbit anti il 6, by Proteintech (1 mentions)
Rabbit anti m opsin, by Merck Group (1 mentions)
Goat anti s opsin, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
6 protocols using anti mif
1
Immunoprecipitation of MIF Signaling Complexes
2
Western Blot Analysis of EMT Markers
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SW480 and HCT116 cells were seeded in 6-well plates until they grew with adherence, the various conditioned media was replaced as described previously and incubated for 24 hours, and cell extracts were prepared in ice-cold lysis buffer containing protease inhibitor. The cell proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride (PVEF) membranes (Millipore). Membranes were further incubated sequentially with specific antibodies including anti-E-cadherin (1:1000, Pro780, Cell Signaling Technology), anti-N-cadherin (1:1000, EPR1791–4, Epitomics), anti-Vimentin (1:1000, EPR3776, Epitomics), anti-MIF (1:1000, Santa Cruz Biotechnology), anti-p-Cofilin (1:1000, 77G2, Cell Signaling Technology), and anti-F-actin (5 μg/ml, 4E3.adl, Abcam). After primary antibodies were incubated, the blots were subsequently incubated with appropriate secondary antibodies. Protein bands were visualized with ECL reagent (Thermo Scientific Inc.) and a Bio-Rad image acquisition system (Bio-Rad Laboratories). The protein bands was quantified using densitometric scanning software, and relative protein abundance was determined by normalization with tubulin or GAPDH.
3
Immunoblot Analysis of Ocular Proteins
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Three to five individual eyecups, without the lens and cornea, from each group were homogenized, centrifuged, and 50 µg of the soluble protein were loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. The proteins were detected with the following primary antibodies: mouse anti-IL-1β (1:1,000, Millipore), rabbit anti-IL-6 (Proteintech, 1:1000), anti-TNF-α (1:1,000, Millipore) and anti-MIF (1:1000, Santa Cruz), mouse anti-rhodopsin (1D4, 1:4,000), rabbit anti-M-opsin (1:1,000, Millipore), goat anti-S-opsin (1:1,000, Santa Cruz), and rabbit anti-caspase 3 (1:1,000, Cell Signaling Technology). After stripping, the same membranes were probed with rabbit anti-actin-HRP (Horseradish peroxidase conjugate; 1:1,000, Cell Signaling Technology) or rabbit anti-GAPDH (1:2,500, Abcam). Development of bands, image capture, and the densitometric analysis of the bands were the same as we previously reported [5 (link)].
4
Western Blot Analysis of CD74, MIF, and DDT
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Tissue and cell samples were homogenized in lysis buffer, separated by 10% or 12% SDS-PAGE under reducing conditions and transferred to PVDF membranes (Millipore, Bedford, MA, USA), blocked with 5% skimmed milk in PBS/0.5% v/v Tween 20 for 1 h, and washed with PBS/Tween [28 (link)]. Primary antibodies were rabbit polyclonal anti-CD74 (1:500, Santa Cruz, CA, USA), anti-MIF (1:500, Santa Cruz, CA, USA) and anti-DDT (1:500, Abcam). Antibodies were diluted in 5% milk PBS/Tween. Blots were washed with PBS/Tween and subsequently incubated with appropriate horseradish peroxidase-conjugated secondary antibody (1:2000, GE Healthcare/Amersham, Aylesbury, UK). After washing, blots were developed with the chemiluminescence method (ECL). Blots were then re-probed with monoclonal anti- mouse α-tubulin antibody (1:2000, Sigma) and levels of expression were corrected for minor differences in loading.
5
Western Blot Analysis of Protein Markers
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Protein lysates were harvested in 9M Urea, 0.075M Tris buffer (pH 7.6) and quantified using BCA assay. Samples were run on SDS-page gels using standard protocol. Antibodies used were: anti-MIF (1:2,500) (Santa Cruz, sc-20121), anti-p53 (1:500) (Santa Cruz; sc-1315), anti-PS15 p53 (1:1,000) (Cell Signaling; 9284S), ABCA1 (1:1000) (Genscript, A00121) and anti- β-Actin (1:50,000) (Santa Cruz; sc-47778). Tumor section staining for MIF were done as previously described [12 (link),17 (link)].
6
Quantitative Immunohistochemistry in Kidney Tissue
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Immunohistochemistry was carried out as previously described on paraffin-embedded 5 μm thick tissue sections [27 (link)]. Primary antibodies were rabbit polyclonal anti-CD74 (1:50, Santa Cruz, CA, USA), anti-DDT (1:100, Abcam) and anti-MIF (1:100, Santa Cruz). Sections were counterstained with Carazzi`s hematoxylin. Negative controls included incubation with a non-specific immunoglobulin of the same isotype as the primary antibody. Sections were subsequently incubated with the proximal tubule marker, fluorescein-conjugated tetragonolobus lotus lectin (1:33, Vector Lab, Peterborough, United Kingdom). Staining was evaluated by a quantitative scoring system, Image-Pro Plus software (Media Cybernetics, Bethesda, MD) in 10 randomly selected fields (x20) per kidney. Samples were examined in a blinded manner.
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