Tumors and cell lysates were prepared as previously described in Ref. [19 (link)]. Total protein extracts (40 μg) were separated under reducing conditions on 5–15% polyacrylamide gels (depending on the molecular weight) and transferred onto polyvinylidene difluoride membranes (NEN, Boston, MA, USA). Membranes were saturated for 1 h with casein (1%, w/v) in PBS-Tween-20 (0.1%, v/v). Antigenic bands were detected by exposing the membranes to human primary antibodies targeting the following proteins: SCD1 [M38], SCD1 [ab52356, Abcam], TxNIP [D5F3E], Cell Signaling (Beverly, MA, USA), FABP4 [ab13979], GPX4 [ab231174] Abcam (Cambridge, England), SOD1 [8B10], Catalase [PA5-23246] Thermofisher (Rockford, IL, USA), GPX1 [A0873] and serotransferrin [A1448] (Abclonal, Woburn, MA, USA). After washes, membranes were incubated with a secondary horseradish peroxydase (HRP)-conjugated goat anti-rabbit antibody (1:2000, DakoCytomation) or sheep anti-mouse antibody (1:1000, DakoCytomation). Immunocomplexes were visualized by chemiluminescence reaction on a luminescent image analyzer (LAS-4000; Fujifilm, Wavre, Belgium). For loading control, membranes were stripped and re-incubated with Hsc70 [B-6, sc-7298] antibody (Santa Cruz, CA, USA), or GAPDH [MAB37A] and actin [A2066] antibody (Sigma Aldrich, Saint-Louis, USA).
Ab52356
Manufactured by Abcam
Sourced in United Kingdom
Ab52356 is a laboratory equipment product manufactured by Abcam. It is designed for general laboratory applications. The core function of this product is to perform a specific task, but a detailed description is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp2 aobs, by Leica (2 mentions)
T8787, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti rabbit antibody, by Agilent Technologies (1 mentions)
Txnip d5f3e, by Cell Signaling Technology (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Ab65407, by Abcam (1 mentions)
Ab12148, by Abcam (1 mentions)
Hrp labelled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Anti flag m2 affinity gel a2220, by Merck Group (1 mentions)
Oligonucleotide, by Merck Group (1 mentions)
Ab108323, by Abcam (1 mentions)
Ab34710, by Cell Signaling Technology (1 mentions)
Protease inhibitors and phosphatase inhibitors, by CWBIO (1 mentions)
Phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Ab2413, by Abcam (1 mentions)
Ripa buffer, by CWBIO (1 mentions)
Ab186735, by Abcam (1 mentions)
Ab207305, by Abcam (1 mentions)
Ab32028, by Abcam (1 mentions)
8 protocols using ab52356
1
Western Blot Analysis of Protein Expression
2
Antibody and Reagent Catalog for Virus Research
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The following antibodies, beads, and dyes were obtained commercially: HCV core (clone 7–50, sc-57800), HA (clone Y-11, sc-805-G), PABPC1 (clone 10E10, sc-32318), MOV10 (clone B-3, sc-515722), calnexin (c-20, sc-6465), TIA1 (c-20, sc-1751) (all Santa Cruz Biotechnology), G3BP1 (Clone 23/G3BP, 611127, BD Biosciences), PLIN2 (GP40, Progen; ab52356, abcam), HCV NS3 (ab65407, abcam), HCV NS5A (HCM-131-5, IBT), FLAG (F7425), FLAG (F1804), HA (H6908), tubulin (clone B5-1-2, T6074), anti-FLAG M2 affinity gel (A2220), anti-HA affinity gel (HA-7, A2095), recombinant protein A (15918–014) and protein G (15920–010) agarose beads (all Sigma), L1ORF1p (clone 4H1, MABC1152, Merck), YB-1 (ab12148, abcam), HRP-labelled secondary antibodies (Jackson ImmunoResearch), HRP-labelled TrueBlot secondary antibodies (Rockland Immunochemicals), Alexa488-, Alexa555-, and Alexa647-conjugated secondary antibodies (all donkey, IgG (H+L)), BODIPY493/503 (D-3922), BODIPY 655/676 (B-3932) (all Life Technologies), Hoechst33342 (Thermo Fisher). L1ORF1p antibody #984 was described previously [33 (link)]. Oligonucleotides and PCR primer were purchased from Sigma. Restriction enzymes for molecular cloning were obtained from NEB, other enzymes from Thermo Fisher. Unless stated otherwise, chemicals were purchased from Sigma or Applichem and cell culture reagents from Gibco/Thermo Fisher.
3
Antibody use in biochemical studies
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The antibodies used in this study are listed as follows: mouse anti‐FLAG (1:3,000; Engibody Biotechnology; AT0022), mouse anti‐HA tag (1:2,000; 6E2; Cell Signaling Technology [CST]; 2367S), mouse anti‐Myc tag (1:2,000; 9B11; CST; 2276S), rabbit anti‐GFP (1:4,000), rabbit anti‐β‐actin (1:1,000; ABclonal; AC026), rabbit anti‐UBR1 (1:1,000; Proteintech; 26069‐1‐AP) for human UBR1 and mouse Ubr1, rabbit anti‐UBR2 (1:1,000; Affinity Biosciences; DF9491) for human UBR2 and mouse Ubr2, rabbit anti‐PLIN2 (1:2,000; Abcam; ab108323) for human PLIN2, rabbit anti‐Plin2 (1:2,000; Abcam; ab52356) for mouse Plin2, and rabbit anti‐Ubr1 (1:2,000) for fly Ubr1.14
4
Protein Extraction and Western Blot Analysis
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RIPA buffer (CWBIO, China) supplemented with protease inhibitors and phosphatase inhibitors (CWBIO, China) was used to extracted protein from kidney tissues or HK-2 cells. Protein concentration was quantified by the BCA method. Equal amounts of protein were then analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Primary antibodies specific for AdipoR1 (1:3000, Abcam, ab126611), phospho-AMPKα(Thr172) (1:1000, Cell Signaling Technology, 2535), AMPKα1 + AMPKα2 (1:2000, Abcam, ab131512), phospho-ULK1 (Ser555) (1:1000, Cell Signaling Technology, 5869), ULK1 (1:1000, Cell Signaling Technology, 8054), ADRP (1:500, Abcam, ab52356), FN (1:1000, Abcam, ab2413), Collagen I (1:1000, Abcam, ab34710), LC3B (1:1000, Cell Signaling Technology, 3868), Beclin1 (1:800, Proteintech, 11306-1-AP), ATG5 (1:1000, Proteintech, 10181-2-AP), SQSTM1/ P62 (1:1000, Proteintech, 18420-1-AP, Cell Signaling Technology, 1:1000, 5114) and TFEB (1:1000, Proteintech, 13372-1-AP) were used. The bands were evaluated using a Tanon 5200 Multi instrument (Tanon Instruments, China). The band densities of target proteins were compared with those of β-actin (1:5000, Proteintech, 60008-1-Ig), GAPDH (1:5000, Proteintech, 60004-1-Ig) or Lamin B (1:1000, Proteintech, 66095-1-Ig) by using densitometry software (ImageJ).
5
Immunostaining of HKC-8 Cells for Cellular Markers
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HKC-8 cells cultured on coverslips or kidney cryosections (3 µm) were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by permeabilizing with 0.2% of Triton X-100 (T8787; Sigma-Aldrich) for 10 min and blocking with 10% of donkey serum for 1 h. Then the slides were immunostained with special primary antibodies overnight at 4°C. The primary antibodies included: anti-fibronectin (F3648; Sigma-Aldrich), anti-γH2AX (ab26350; Abcam), anti-TOMM20 (ab186735; Abcam), anti-mTOR (ab32028; Abcam), anti-P62 (ab207305; Abcam), anti-ADFP (ADRP) (ab52356; Abcam), anti-active β-catenin (19807s, Cell Signaling Technology) and anti-LC3B (ab48394; Abcam). The next day, the slides were stained with a Cy3-or Cy2-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h, followed by DAPI (Sigma-Aldrich) staining for 10 min. Finally, Images were taken by confocal microscopy (Leica TCS SP2 AOBS, Leica Microsystems, Buffalo Grove, IL).
6
Immunostaining of HK-2 cells and mouse kidney
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HK‐2 cells cultured on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by permeabilizing with 0.2% of Triton X‐100 (T8787; Sigma‐Aldrich) for 10 min and blocking with 10% of donkey serum for 1 h. Paraffin‐embedded mouse kidney sections (3 μm thickness) were prepared by a routine procedure. Then the slides were immunostained with special primary antibodies overnight at 4°C. The primary antibodies included: anti‐fibronectin (F3648; Sigma‐Aldrich), anti‐Flag (M185‐3S, MBL), anti‐ADRP (ab52356; Abcam), β‐catenin (ab15180; abcam), anti‐CXCR4 (sc‐53,534; Santa), anti‐CPT1A (ab128568; abcam), anti‐Lotus Tetragonolobus Lectin (LTL) (FL‐1321; VECTOR Laboratories), anti‐Peanut Agglutinin (PNA) (FL‐1071; VECTOR Laboratories) and anti‐Aquaporin 3 (AQP3) (ab125219; abcam). After washing, slides were incubated with Cy2‐ or Cy3‐conjugated donkey anti‐mouse or anti‐rabbit IgG (Jackson Immuno‐Research Laboratories, West Grove, PA). Nuclei were stained with DAPI (C1006, Beyotime) according to the manufacturer's instructions. Images were taken by confocal microscopy (Leica TCS SP2 AOBS, Leica Microsystems, Buffalo Grove, IL).
7
Western Blot Analysis of Antioxidant Proteins
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Cells were harvested and lysed. BCA protein assay kit was used to measure the concentration of proteins. Equal amount of denatured proteins (35 μg) were loaded into corresponding lanes, separated by 12% SDS-polyacrylamide gel electrophoresis (Beyotime, China) and then electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Germany). The membranes were blocked with 5% non-fat dried milk in TBST containing 1% Tween-20 for 2 h at room temperature, and then incubated with the primary antibodies overnight at 4°C. On the second day, the membranes were incubated with secondary antibodies, then washed. After the incubation with enhanced chemiluminescence reagents (Beyotime, China), the blots were detected by using MicroChemi™ Chemiluminescent Imaging System (DNR Bio-Imaging Systems, Israel). The result was quantified by using Image-Pro Plus 6.0. All values were normalized to the internal control of GAPDH. All experiments were performed at least three times.
Primary antibodies included anti-GAPDH (60004-1-1g, Proteintech, China), anti-Nrf2 (ab137550, Abcam, UK), anti-HO-1 (5853S, Cell Signaling Technology, USA) anti-NQ-O1 (ab28947, Abcam, UK), and anti-PLIN2 (ab52356, Abcam, UK) antibodies. The secondary antibody was a HRP-conjugated goat anti-rabbit or an anti-mouse IgG polyclonal antibody (Beyotime, China).
Primary antibodies included anti-GAPDH (60004-1-1g, Proteintech, China), anti-Nrf2 (ab137550, Abcam, UK), anti-HO-1 (5853S, Cell Signaling Technology, USA) anti-NQ-O1 (ab28947, Abcam, UK), and anti-PLIN2 (ab52356, Abcam, UK) antibodies. The secondary antibody was a HRP-conjugated goat anti-rabbit or an anti-mouse IgG polyclonal antibody (Beyotime, China).
8
Immunofluorescence Staining of Liver Sections
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Liver sections were incubated with the indicated primary antibodies, such as anti-ABCD3 (1:200, ab85550, Abcam, Cambridge, UK), anti-catalase (1:200, sc271803, Santa Cruz Biotechnology Inc.), and anti-adipose differentiation-related protein (ADFP, 1:200, ab52356, Abcam). After incubation with the primary antibodies, the liver sections were subsequently incubated with Alexa 488-conjugated goat anti-mouse (1:1,000, A11018, Invitrogen, Carlsbad, CA, USA) and Alexa 568-conjugated goat anti-rabbit (1:1,000, A11070, Invitrogen). 4´,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1:1,000, 62248, Thermo Fisher Scientific, Waltham, MA, USA) was used for cell nuclei staining.
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