For Western analysis, immunoprecipitated nuclear extracts were separated by 10% sodium dodecyl sulfate (SDS)-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon FL, Millipore). The following primary antibodies were used for immunoblotting: anti-REST antibody (Millipore, 17–641), anti-HDAC1 (Abcam, ab7028), anti-HDAC2 (Abcam, ab51832), anti-LSD1 (Abcam, ab17721), anti-NME2 (Abcam, ab60602) (23 (link), 24 (link), 40 (link)), and anti-hTERT (Abcam, ab32020) (49 (link), 50 ). The secondary antibodies used were anti-mouse and anti-rabbit alkaline phosphatase conjugates from Sigma. Western blots with antibodies against NME2, telomerase, REST, and LSD1 along with relevant molecular weight markers are shown in supplemental Fig. S5 .
Ab32020
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab32020 is a laboratory equipment product. It is used for specific scientific applications. No further details are available to provide an unbiased and factual description while maintaining conciseness.
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Lab products found in correlation
Ab17721, by Abcam (2 mentions)
Abx120550, by Abbexa (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab7028, by Abcam (1 mentions)
Ab51832, by Abcam (1 mentions)
Ab60602, by Abcam (1 mentions)
Immobilon fl, by Merck Group (1 mentions)
Anti mouse and anti rabbit alkaline phosphatase conjugates, by Merck Group (1 mentions)
Nb100 68252, by Abcam (1 mentions)
Nb300 269, by Novus Biologicals (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Observer z1, by Zeiss (1 mentions)
Alexa fluor 594 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Super signal western pico chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
A5441, by Merck Group (1 mentions)
A8592, by Merck Group (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Fluorchem hd2 imaging system, by Bio-Techne (1 mentions)
20 protocols using ab32020
1
Western Blot Analysis of Nuclear Proteins
2
Phosphorylation-Specific Antibody Development
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Anti-hTERT mouse monoclonal antibodies (mAbs, clones 10E9-2 and 2E4-2) were generated and described the specificity as reported previously14 (link). Phosphospecific rabbit polyclonal antibodies (pAbs) and mouse mAb against phosphorylated threonine 249 of hTERT (anti-249T-P and TpMab-1, respectively) were generated in this study (see below). Anti-hTERT mouse mAb (1:100, clone 2E4-2), anti-hTERT rabbit mAb (1:1000, Abcam, ab32020), anti-249T-P pAbs (1:1000), TpMab-1 mAb (1:50), anti-phospho-histone H3 (Ser10) rabbit pAbs (1:2000, Merck, 06-570), anti-cdc2 [CDK1] (POH1) mouse mAb (1:1000, Cell Signaling Technology, 9116), anti-β-actin (AC-15) mouse mAb (1:20000, Sigma-Aldrich, A5441), anti-FOXO4 rabbit mAb (1:4000, Abcam, ab128908), anti-GAPDH mouse mAb (1:1000, MBL Co., Ltd., M171-3), and anti-SMG6 rabbit pAbs64 (link) (1:1000) were used for immunoblotting (IB). Anti-hTERT mAb (clone 10E9-2: MBL Co., Ltd., M216-3) and anti-hTERT sheep pAbs (Abbexa Ltd., abx120550) were used for immunoprecipitation (IP). Anti-CDK1 rabbit pAbs (1:250, Merck, HPA003387), anti-249T-P pAbs (1:250) and TpMab-1 mAb (1:250) were used for immunohistochemistry.
3
Western Blot Analysis of Telomerase Proteins
4
Confirming hTERT Protein Expression via Immunofluorescence
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To confirm transgenic hTERT protein expression, we performed immunofluorescence (IF) staining on the hTERT- immortalized cell lines as well as non-transfected fibroblasts and ES cells as controls. Prior to the beginning of the procedure, the subjected cells were detached with TrypLE and seeded in 48-well plates. After reaching an appropriate density, the cells were fixed in 4% PFA (in PBS) for 10–30 min and permeabilized with 0.1% TritonX-100 for 10–15 min at room temperature. Following double wash with PBS, the cells were incubated with primary antibody for hTERT (Abcam, Cat. # ab32020; diluted 1:200 in PBS + 5% BSA) overnight at 4°C. Afterwards, the cells were washed twice with PBS and incubated with the secondary antibody, AlexaFluor594 donkey anti-rabbit (Life Technologies, #A21207; diluted 1:200 in PBS + 5% BSA) for 20 min at room temperature in the dark and subsequently stained with 5% DAPI in PBS for 1–2 min at room temperature. Finally, the cells were washed twice with PBS and mounted with Citifluor mountant medium (CITIFLUOR). Microscopy images were taken with a Zeiss Observer Z1 (Zeiss).
5
Western Blot Protein Analysis Protocol
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Western blots were carried out using 4–12% polyacrylamide Bis Tris Glycine gels (Life Technolgies) and antibodies against hTERT (Ab32020; 1:1000; Abcam, UK), beta-Actin (A5441; 1:5000; Sigma), and an HRP-conjugated FLAG-antibody (A8592; 1:1000; Sigma). Secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used at 1:5000. Detection was carried out using SuperSignal Western Pico Chemiluminescence substrate (ThermoFisher Scientific, Waltham, MA).
6
Western Blot Analysis of TERT
7
Immunoprecipitation and qPCR Analysis of hTERT
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Hs68 and Hs68 + hTERT cell lysates were used for FLAG-hTERT immunoprecipitation as described elsewhere59 (link). Briefly, NP-40 lysis buffer (10 mM Tris-HCl pH 7.5, 1% (v/v) NP-40, 10% (v/v) glycerol, 1 mM EGTA, 1 mM MgCl2, 150 mM NaCl) containing Protease Inhibitor Cocktail tablet (Roche) was used to lyse the cells and anti-FLAG M2 affinity resin (Sigma-Aldrich, Canada) was used to immunoprecipitate FLAG-hTERT. After incubation, proteins were eluted from the beads using SDS buffer. Flag-hTERT immunoprecipitates from Hs68 cells were detected via western blotting using rabbit monoclonal (Y182) to telomerase reverse transcriptase (1:1000; ab32020; Abcam) as previously described62 (link). For qPCR, RNA was isolated using TRizol Reagent (Invitrogen) using the manufacturer's protocol. After incubation with chloroform and isopropanol, the precipitated RNA was washed with 70% ethanol and air dried before disolving in RNAse-free ddH2O. cDNA was made using 2 µg RNA using qScript cDNA SuperMix reaction protocol (Quantabio). Specific primers for hTERT and GAPDH (house keeping gene) were used to determine the relative hTERT levels in Hs68 and Hs68 + hTERT cells.
8
Western Blot Validation of Protein Expression
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Western blots were performed to verify the expression of proteins in control and IPF group. Total protein extracts (20 μg) were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with anti-KLF4 (Abcam, Ab214666), anti-TERT (Abcam, Ab32020) and anti-GAPDH (Abcam, Ab8245) antibodies. The signal was detected by enhanced automatic chemiluminescence camera (Tanon 5200).
9
Western Blot Analysis of Protein Expression
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Western blot was performed as described elsewhere. Briefly, the fresh cells were harvested, washed with PBS, and lysed in lysis buffer. After being boiled, the proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes. Primary antibodies used were as follows: anti-TERT (1 : 1000 diluted, ab32020, Abcam), β-catenin (1 : 1000 diluted, 8480, Cell Signaling Technology, USA), c-Myc (1 : 1000 diluted, ab32072, Abcam), and GAPDH (1 : 1000 diluted; 5174, CST). Next day, proteins were incubated with horseradish peroxidase-conjugated secondary antibody and visualized by using the enhanced chemiluminescence method. The intensity of the bands was quantified using the image lab 6.0 system (Bio-Rad Laboratories, USA), and the data were normalized to the GAPDH as loading controls. All western immunoblot analyses were performed three times.
10
Evaluating LDHB Expression in Pancreatic Cancer
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Paraffin specimens of pancreatic cancer tissue were obtained from surgically removed tissues of inpatients in the Faculty of Hepato-Pancreato-Biliary Surgery, Chinese PLA General Hospital. The ethics of the study was approved by the Institutional Review Board of Chinese PLA General Hospital (the reference number is S2016-098-01). IHC of formalin-fixed paraffin-embedded samples was performed as described previously (18 (link)). Rabbit anti-LDHB (ab32020, Abcam, USA) was used at a dilution of 1:150. The expression of LDHB was determined by calculating the expression score which was generated by multiplying the score for intensity of the staining (no staining=0; weak staining=1; moderate staining=2; strong staining=3) by the percentage of stained cells (0%-100%). Expression score above 1.6 was determined as high expression and below 1.6 was set as low expression.
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