Glyceraldehyde 3-phosphate dehydrogenase (60004-1-Ig) and horseradish peroxidase-conjugated secondary antibodies (SA00001-1 and SA00001-2) were purchased from Proteintech (Wuhan, China). Antibodies against Lamp-2a (ab125068), HSP90 (ab32568), GPX4 (ab252833), HSC70(ab51052), and 4-HNE (ab46545) were obtained from Abcam (Cambridge, UK). NVP-AUY922 and ferrostatin-1 (Fer-1) were purchased from Selleck Chemicals (Houston, TX, USA).
Ab51052
Manufactured by Abcam
Sourced in United Kingdom
Ab51052 is a recombinant monoclonal antibody that recognizes the protein CD3 epsilon. It is designed for use in various immunoassays and applications involving the detection and study of CD3 epsilon.
Automatically generated - may contain errors
Lab products found in correlation
Ab125068, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab252833, by Abcam (1 mentions)
Nvp auy922, by Selleck Chemicals (1 mentions)
Ferrostatin 1 fer 1, by Selleck Chemicals (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Ab46545, by Abcam (1 mentions)
Hrp conjugated antibodies, by Agilent Technologies (1 mentions)
α sma 14 9760 82, by Thermo Fisher Scientific (1 mentions)
Anti mouse cy3, by Merck Group (1 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Gene Tech (1 mentions)
Ab22595, by Abcam (1 mentions)
Ab13524, by Abcam (1 mentions)
Ab2900, by Abcam (1 mentions)
Ab217345, by Abcam (1 mentions)
Mabt837, by Merck Group (1 mentions)
Ab78078, by Abcam (1 mentions)
10 protocols using ab51052
1
Antibody-based Analysis of Cellular Stress Pathways
2
Western Blot Antibody Validation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies against Furin (70393), Vimentin (5741), N-cadherin (14215), and β-Actin (4970) were purchased from Cell Signalling Technology (Danvers, MA, USA). Those against β-Catenin (ab32572), hsc70 (ab51052), and GAPDH (ab8245) were from Abcam (Cambridge, UK). Other primary antibodies used were: E-cadherin (80182) from BD Biosciences (Bedford, MA, USA) and α-SMA (14-9760-82) from Invitrogen™ (Waltham, MA, USA). All the HRP-conjugated antibodies were from DAKO (Nowy Sącz, Poland) and for immunofluorescence detection Alexa Fluor 488 anti-rabbit IgG (Invitrogen™) and Cy3 anti-mouse (Sigma-Aldrich, St. Louis, MO, USA) were used. For the ChIP assay, antibodies against RARα (sc-515796) and non-specific IgG (sc-2025) were used, both from Santa Cruz Biotechnology (Dallas, TX, USA).
3
Immunohistochemical Evaluation of HSC70 in Renal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Resected renal cancer specimens were fixed in 4% formaldehyde and embedded in paraffin. Cancer tissue samples, which were sliced into 5-μm‒thick sections and placed on glass slides treated with 3-aminopropyltriethoxysilane, were deparaffinized and rehydrated. After that, they were incubated overnight at 4 °C with a primary antibody against HSC70 (1:50) purchased from Abcam (ab51052). After washing, cells probed with the primary HSC70 antibody were detected by incubating with horseradish peroxidase (HRP)-conjugated secondary antibody at 37 °C for 45 min (purchased from Gene Tech, Shanghai), and then with DAB (DAB kit; purchased from Gene Tech, Shanghai). The primary antibody was substituted with phosphate-buffered saline in the negative control. Immunohistochemical signals were evaluated independently by two pathologists.
The expression of HSC70 was semi-quantitatively classified according to the following criteria: a score of 0 if <1% of cancer cells expressed nuclear and/or cytoplasmic HSC70; 1+ if ≥1% and <5% of cancer cells expressed nuclear and/or cytoplasmic HSC70; 2+ if ≥5% and <10% of morphologically unequivocal cancer cells expressed nuclear and/or cytoplasmic HSC70; and 3+ if ≥10% were positive cells; 2+ and 3+ were considered HSC70-positive.
The expression of HSC70 was semi-quantitatively classified according to the following criteria: a score of 0 if <1% of cancer cells expressed nuclear and/or cytoplasmic HSC70; 1+ if ≥1% and <5% of cancer cells expressed nuclear and/or cytoplasmic HSC70; 2+ if ≥5% and <10% of morphologically unequivocal cancer cells expressed nuclear and/or cytoplasmic HSC70; and 3+ if ≥10% were positive cells; 2+ and 3+ were considered HSC70-positive.
4
Immunoblotting for Exosome Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following primary antibodies were used: rabbit polyclonal anti-KIBRA (sc-133374; Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-KIBRA (clone 2A5, provided by Jixin Dong’s lab); mouse monoclonal anti-Alix (#2171; Cell Signaling Technology, Danvers, MA, USA); mouse monoclonal anti-Tsg101 (ab83; Abcam, Cambridge, MA, USA); mouse monoclonal anti-CD63 (ab217345; Abcam); rabbit polyclonal anti-calnexin (ab22595; Abcam); mouse monoclonal anti-LBPA (MABT837; Sigma St. Louis, MO, USA); rabbit polyclonal anti-EEA1 (ab2900; Abcam); rabbit polyclonal anti-LC3B (#2775; Cell Signaling Technology); rat monoclonal anti-LAMP2 (ab13524; Abcam); rabbit monoclonal anti-Rab27a (#69295; Cell Signaling Technology); mouse monoclonal anti-beta III Tubulin (ab78078; Abcam); and rabbit monoclonal anti-Hsc70 (ab51052; Abcam). CHX (#2112) was purchased from Cell Signaling Technology; Baf (A8510) was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China), and Lac (ab141411) was purchased from Abcam. When indicated, the medium contained 40 μg/ml CHX or 20 nM Baf or 10 μM Lac.
5
Disaggregation of α-Synuclein Oligomers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
α-Syn disaggregation was carried out in disaggregation buffer at final protein concentrations of 10 µM α-syn, 10 µM Hsc70, 5 µM DnaJB1 and 1 µM Apg2 in a total volume of 400 µL in the presence of ATP (2 mM) and an ATP-regeneration system (8 mM phosphoenol pyruvate and 20 ng/µL pyruvate kinase). After incubation (2.5 h at 30 °C), the reaction mixture was applied to 3.2 mL of a 5–40 % sucrose gradient. In the case of α-syn oligomers, sample and gradient volumes were halved to save protein. Samples were centrifuged at 162,000 g for 2 h at 4 °C and 400 µL fractions (200 µL for oligomers) were manually removed and subjected to SDS-PAGE and immunoblotting using an anti-α-syn antibody (Invitrogen PA5-85343, 1:2000 dilution). Alternatively, samples were analyzed using antibodies against Hsc70 (Abcam ab51052; 1:5000), Apg2 (Abcam ab185962; 1:5000) and DnaJB1 (Enzo ADI-SPA-450, 1:2000).
6
Immunoblot Analysis of Bladder Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibodies used in this study included anti‐HSC70 (ab51052; Abcam), anti‐caspase 3 (ab32351; Abcam) and anti‐beta actin (ab8226; Abcam). First, the proteins of bladder cancer cells were extracted, and the protein concentration was determined. Then, the proteins of each group were electrophoresed under the conditions of a constant pressure of 100 V and stopped when the dye front moved 2–3 mm from the bottom of the gel (approximately 120 min). Subsequently, we transferred the proteins on the gel to the FVDF membrane under the following conditions: constant current 200 mA for 2 h. Next, we added 5% skim milk to the PVDF membrane containing protein and blocked it at room temperature for 1 h. The sealed PVDF membrane was incubated with primary antibody and then placed in a 4°C refrigerator overnight. The next day, the primary antibody was recovered, and the secondary antibody was added and incubated at room temperature for 1 h. Scanning densitometry was performed with ImageJ software (V1.53), and GraphPad Prism software (V9.4.1) was used to generate statistical graphs.
7
Immunoblotting of Oxidized DJ-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Separated proteins were transferred to Hybond P membrane (GE Healthcare). BN PAGE membranes were destained with methanol and stained with Ponceau to mark protein ladder (Native Mark Unstained Protein Standard, Life Technologies) and check for equal protein loading. Membranes were blocked for 30 min at room temperature in 10% milk in PBS and subsequently probed with monoclonal mouse anti-total DJ-1 (3E8, Enzo Life Sciences; 1:5,000), monoclonal mouse anti-oxidised DJ-1 (clone M106, kind gift from Yoshiro Saito; 1:1,000), anti-hnRNPA1 (abcam ab50966; 1:1000), anti-GAPDH (abcam ab8245; 1:40,000), anti-hsc70 (abcam ab51052), anti-PABP1 (abcam ab21060; 1:50) and anti-β-actin (abcam ab82618; 1:50,000). After washing with PBS/0.4% (v/v) Tween20 (PBS/T), blots were incubated with respective horse radish peroxidase-coupled secondary antibodies (Dako). Blots were developed with either in-house chemiluminescence solution or enhanced chemiluminescence kit (Pierce). Densitometry of bands was quantified using imageJ (NIH).
8
Western Blot Antibodies and Reagents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit monoclonal anti-CD9 antibody (EPR2949, ab92726; Abcam, Shanghai, China), rabbit monoclonal anti-Hsc70 antibody (EP1531Y, ab51052; Abcam, Shanghai, China), and rabbit monoclonal anti-ER alpha antibody (E115, ab32063; Abcam, Shanghai, China) were used in this study. Normal rabbit IgG was obtained from R&D Systems (Minneapolis, MN, USA). Filtered and sterile phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). A pre-stained protein ladder was obtained from Thermo Fisher Scientific (Baltics UAB, Vilnius, Lithuania).
9
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer (Beyotime) and the supernatant was taken for determination of protein concentration with bicinchoninic acid kits (Boster). Protein was put to loading buffer, denatured via a 10-min boiling water bath, and electrophoresed. Separated protein bands were moved onto polyvinylidene fluoride membranes, which were immersed in 5% non-fat powdered milk blocking buffer at ambient temperature for 1 h, followed by overnight incubation at 4°C with HSP90 (1:500, ab59459), HSC70 (1:2,000, ab51052), aLAMP-2A (1:2,000, ab125068), Snail (1:1,000, ab216347), E-cadherin (1:1,000, ab1416), and GAPDH (1:1,000, ab8245) antibodies (all from Abcam). After three washes with TBST (10 min each), the membranes were probed with HRP-labeled IgG antibodies and then treated with enhanced chemiluminescence reagents (P0018FS; Beyotime). The blots were imaged with a Bio-Rad chemiluminescence imaging system and analyzed using Quantity One v4.6.2 software.
10
Mitochondrial Protein Interactome in PD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A mitochondrial PPI network was generated by retaining interactions from two or more published studies (PMID are listed in Supplementary Table 5 for each interaction) for proteins whose level was altered in PARK2-mutated patients. The network was then visualized using Cytoscape.
A small set of interesting candidate interactions of physiological relevance was confirmed in control subject C4 and PARK2-mutated PD patient P3 by immunoprecipitation. Mitochondrial pellets were cross-linked with 1 mM dithiobis (succinimidyl propionate) (DSP; Thermo Fisher Scientific) and lysed with 500 μl RIPA buffer. Inputs were controlled by western blotting. Three microliter of each antibody [HSPA8, abcam (ab51052); HSPD1, abcam (ab46798); ALDH2, Santa Cruz Biotechnology (sc-100496)] were added to 1 mg of mitochondrial protein lysates, which were then incubated for 1 h in agitation at 4°C. One hundred microliter of μMACS magnetic microbeads (Miltenyi) were then added to lysates and incubated with continued agitation ON at 4°C. After washing the microbeads suspensions using 0.1% RIPA buffer through μMACS columns (Miltenyi), proteins were eluted using 200 μl of Laemmli buffer, previously heated at 95°C. Eluates were analyzed by western blotting.
A small set of interesting candidate interactions of physiological relevance was confirmed in control subject C4 and PARK2-mutated PD patient P3 by immunoprecipitation. Mitochondrial pellets were cross-linked with 1 mM dithiobis (succinimidyl propionate) (DSP; Thermo Fisher Scientific) and lysed with 500 μl RIPA buffer. Inputs were controlled by western blotting. Three microliter of each antibody [HSPA8, abcam (ab51052); HSPD1, abcam (ab46798); ALDH2, Santa Cruz Biotechnology (sc-100496)] were added to 1 mg of mitochondrial protein lysates, which were then incubated for 1 h in agitation at 4°C. One hundred microliter of μMACS magnetic microbeads (Miltenyi) were then added to lysates and incubated with continued agitation ON at 4°C. After washing the microbeads suspensions using 0.1% RIPA buffer through μMACS columns (Miltenyi), proteins were eluted using 200 μl of Laemmli buffer, previously heated at 95°C. Eluates were analyzed by western blotting.
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!