The transfected MDA-MB-231 and HS598T cells were treated with RIPA lysis buffer supplemented with protease inhibitor cocktail tablet and phosphatase inhibitor for protein extraction. Afterward, cells were centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was reserved and employed for protein measurement. Then, 20 μg of protein was run on 7.5–12.5% SDS-PAGE gel, and electro-transferred to PVDF membrane. Once blocked with 0.5% skim milk powder, the membrane was incubated at 4 °C with Myc primary antibody (ab39688; Abcam). Then, the anti-Rabbit IgG was added and incubated for 1 h at room temperature. The Novex® ECL HRP Chemiluminescent Substrate Reagent Kit (Invitrogen) was employed for band visualization. For IHC staining, the paraffin-embedded TNBC patient and xenograft tumor tissues were dewaxed by xylene, blocked by H2O2 and BSA, incubated with above Myc primary antibody (ab39688; Abcam), and visualized with DAB solution. Lastly, the tissue slices were sealed by neutral gum. The staining result was quantified by the H-score method as previously described.14 (link)
Ab39688
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab39688 is an antibody-based product manufactured by Abcam. It is designed for use in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab226977, by Abcam (6 mentions)
Ab32572, by Abcam (6 mentions)
Ab16051, by Abcam (5 mentions)
Ab6721, by Abcam (5 mentions)
Ab16663, by Abcam (4 mentions)
Ab181602, by Abcam (4 mentions)
Ab134175, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ab9485, by Abcam (3 mentions)
Ab131442, by Abcam (3 mentions)
Ab1416, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Ab8245, by Abcam (2 mentions)
Ab38898, by Abcam (2 mentions)
Ab155549, by Abcam (2 mentions)
Ab199396, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit, by Santa Cruz Biotechnology (2 mentions)
38 protocols using ab39688
1
Quantification of Myc Protein in TNBC
2
Protein Extraction and Analysis Protocol
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After treatment, the cells were scraped off, transferred into a 2.5 ml EP tube and added with 150 µl mixture of radioimmunoprecipitation assay (RIPA) and protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA), followed by ultrasonic dispersion using an ultrasonic apparatus (40 A, 3 sec/time, repeated 3 times). After centrifugation at 12,000 × g at 4°C for 15 min, the supernatant was taken as the tissue protein. The protein concentration was measured using the bicinchoninic acid (BCA) kit (Beyotime Institute of Biotechnoogy). After denaturation, the total protein was separated using 10% acrylamide gel, transferred onto a 0.22 µm nitrocellulose membrane (EMD Millipore) for 1.5 h, sealed with 5% skim milk for 1 h and incubated with rabbit anti-human fibulin-2, β-catenin, cyclin D1, C-myc and GAPDH polyclonal antibodies (cat. nos. ab251662, ab16051, ab226977, ab39688 and ab9485, respectively; Abcam; diluted at 1:1,000) overnight. Then the band was incubated with the goat anti-rabbit IgG secondary polyclonal antibody (cat. no. ab6721; Abcam; diluted at 1:300) for 1 h, and the target protein band was developed using the ECL system (Bio-Rad Laboratories). The relative content of the target protein was calculated as: gray value (target protein)/gray value (corresponding internal reference band).
3
Immunoblotting Analysis of Protein Expression
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Whole protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime) and quantified using the bicinchoninic acid Protein Assay Kit (Beyotime). Briefly, a 20 μg protein aliquot was loaded into 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for separation, followed by electroblotting on a polyvinylidene fluoride membrane (Merck Millipore, Burlington, MA, USA), blocking using 5% fat-free milk, and overnight incubation with the following primary antibodies: anti-UBR5 (ab70311; Abcam), anti-AXIN1 (ab55906; Abcam), anti-β-catenin (ab32572; Abcam), anti-Survivin (ab76424; Abcam), anti-C-myc (ab39688; Abcam), anti-lactate dehydrogenase A (LDHA, ab101562; Abcam), anti-pyruvate kinase isozymes M2 (PKM2, ab137852; Abcam), anti-H3 (ab1791; Abcam), and anti-GAPDH antibody (60004-1-1G; ProteinTech, Rosemont, IL, USA). They were washed thrice with Tris-buffered saline with Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies (A0208, A0216; Beyotime). Finally, the blot was imaged using the Enhanced Chemiluminescence Detection Kit (Pierce Biotechnology), and the band intensity was measured using Image-Pro Plus 6.0 software, with GAPDH as the loading control.
4
Immunohistochemistry Protocol for METTL8, PCNA, C-myc, and Cdc25C
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Immunohistochemistry (IHC) was performed with a two-step detection kit (Zsbio PV73 9000, China). The paraffin-embedded tissue sections were dewaxed in xylene, rehydrated in a graded alcohol system, and boiled in high-pressure autoclaved citric acid buffer (pH 6.0) for 15 min, and the peroxidase activity was quenched with 3% hydrogen peroxide for 20 min to avoid non-specific staining. The sections were washed three times with PBS followed by incubation overnight with anti-METTL8 antibody (Abcam, ab177201 at 1/200 dilution), PCNA antibody (Abcam, ab265609 at 1/200 dilution), C-myc antibody (Abcam, ab39688 at 1/100 dilution), or Cdc25C antibody (Abcam, ab32444 at 1/200 dilution) at 4°C. After that step, the sections were washed with PBS three times and incubated at room temperature for approximately 20 min with a reaction enhancer kit. This step was followed by three washes in PBS, incubation with secondary antibody at room temperature for 20 min, and staining with 3,3-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China). The sections were dehydrated and sealed after redyeing with hematoxylin.
5
Western Blot Analysis of Glial and Inflammatory Markers
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Western blot analysis was performed as previously described [10 (link)] using the following primary antibodies: rabbit polyclonal anti-GFAP (ab7260, 1:1000), mouse monoclonal anti-OX42 (ab1211, 1:500), rabbit polyclonal anti-interleukin (IL)-1β (ab9722; 1:500), mouse monoclonal anti-IL-6 (ab9324; 1:500), rabbit polyclonal anti-tumor necrosis factor (TNF)-α (ab6671; 1:500), rabbit monoclonal anti-p-Stat3 (ab76315; 1:500), and rabbit polyclonal anti-c-Myc (ab39688; 1:500) (all from Abcam, Cambridge, MA, USA). Rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Abcam, ab181602, 1:3000) was used as a control. Protein band density was quantified using an Epson V330 Photo scanner (Seiko Epson Co., Nagano, Japan) and Quantity One software (Bio-Rad, Hercules, CA, USA).
6
Western Blot Analysis of GBM Pathways
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Protein expressions of AEBP1, phosphorylated IκBα, IκBα, phosphorylated NF-κB p65, NF-κB p65, Cyclin D1 (CCND1), MYC Proto-Oncogene (c-Myc), Matrix Metallopeptidase 9 (MMP9), and Snail Family Transcriptional Repressor 2 (Slug) were detected by western blot. GBM cells were lysed with RIPA buffer containing protease inhibitors. Total proteins were separated by electrophoresis in 10% SDS-PAGE and subsequently transferred to PVDF membranes. The membranes were then incubated with primary antibodies overnight at 4°C, followed by an incubation with secondary antibodies for 1 h at room temperature. Immunoreactive bands were revealed by chemiluminescence, and relative expression of the target protein was normalized to that of GAPDH used as an internal reference. The primary antibodies used in this study were purchased from Abcam (USA): anti-AEBP1 (ab168355), anti-IκBα (ab7217), anti-p-IκBα (ab133462), anti-NF-κB p65 (ab16502), anti-p-NF-κB p65 (ab86299), anti-CCND1 (ab16663), anti-c-Myc (ab39688), anti-MMP9 (ab38898), anti-Slug (ab27568), and anti-GAPDH (ab8245).
7
Protein Expression Analysis in HeLa Cells
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RIPA protein extraction reagent (Beyotime, Shanghai, China) with PMSF (Roche, Basel, Switzerland) was used to lysed HeLa cells. 10% SDS-PAGE was used to separate the protein and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). 5% non-fat milk was used to block the membranes for 2 h. The membranes were probed with anti-GSPT1 (Abcam, ab126090), anti-WNT3A (Abcam, ab28472), anti-ICAM1 (Abcam, ab222736), anti-Vimentin (Abcam, ab8069), anti-E-cadherin (Abcam, ab53013), anti-p-β-catenin (Abcam, ab11350), β-catenin (Abcam,ab32572), anti-c-myc (Abcam, ab39688), anti-cyclin D1 (Abcam, ab226977), anti-GAPDH (Abcam, ab181602) antibodies overnight at 4˚C. Subsequently, membranes were incubated with a HRP-conjugated secondary antibody (CST, #7074) for 1 h at 37˚C. Finally, the blots were visualized by ECL (Thermo Fisher Scientific) and detected using a ChemiDoc XRS imaging system. Band densities were analyzed using Image J software (National Institute of Health, Bethesda, MD, USA). Relative protein levels were determined by normalizing the densitometry value of the proteins of interest to that of GAPDH.
8
Western Blot Analysis of Protein Expression
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Total protein was first extracted using SDS-PAGE and then transferred to a PVDF membrane (Thermo, USA). Afterwards, the PVDF membrane was incubated with 1:1000 dilution of antibody: TRIM47 (ab155549, Abcam, USA), BAP1 (ab199396, Abcam, USA), P21 (ab188224, Abcam, USA), P53 (ab131442, Abcam, USA), SRC (ab47405, Abcam, USA), MET (ab51067, Abcam, USA), and c-Myc (ab39688,Abcam, USA). The membrane was washed and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated goat anti-rabbit (Santa Cruz, USA). SuperSignalMT West Puico PLUS (Termo Scientific, USA) was used to develop the blot, using b-actin (ab8226, Abcam, USA) as the loading control. All experiments were performed in triplicate.
9
Western Blot Analysis of Myc, MAPK, and JNK
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After treatment, H9c2 cells were lysed under the support of RIPA buffer accompanied by protease inhibitor. After isolation by SDS-polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. Next, after blocking by 5% defatted milk, the membranes were incubated with primary antibodies (Abcam, U.K.) against Myc (ab39688, 1:1000), p-P38 MAPK (ab4822, 1:1000), P38 MAPK (ab31828, 1:1000), p-JNK (ab124956, 1:3000), JNK (ab179461, 1:1000), and GAPDH (ab181602, 1:5000) at 4°C for 24 h. After washing with PBS, the membranes were incubated with secondary antibodies at 25°C for another 2 h. Lastly, the signals of protein bands were developed with enhanced chemiluminescence (ECL) and quantified with ImageJ software.
10
Western Blot Analysis of Protein Expression
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RKO cells were harvested and their proteins were extracted by using lysis buffer.
The primary antibodies against CEMIP (DF12056, Rabbit polyclonal antibody;
Affinity Biosciences), GAPDH (ab128915; Abcam, Cambridge, MA, USA), c-Myc
(ab39688; Abcam), E-cadherin (ab1416; Abcam), and Twist-1 (ab49254; Abcam) were
diluted at a ratio of 1:1000, and the second antibody goat anti-Rabbit IgG
H&L (HRP; ab6721; Abcam) was diluted at a ratio of 1:10,000 according to the
instructions. The detailed process for Western blot analysis was conducted as
previously described.15 (link)
The primary antibodies against CEMIP (DF12056, Rabbit polyclonal antibody;
Affinity Biosciences), GAPDH (ab128915; Abcam, Cambridge, MA, USA), c-Myc
(ab39688; Abcam), E-cadherin (ab1416; Abcam), and Twist-1 (ab49254; Abcam) were
diluted at a ratio of 1:1000, and the second antibody goat anti-Rabbit IgG
H&L (HRP; ab6721; Abcam) was diluted at a ratio of 1:10,000 according to the
instructions. The detailed process for Western blot analysis was conducted as
previously described.15 (link)
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