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.
Ab137852
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab137852 is a lab equipment product. It is a device used for scientific research and analysis in a laboratory setting. The core function of this product is to perform specific tasks required for experimental procedures. No further details can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab115730, by Abcam (5 mentions)
Ab209847, by Abcam (5 mentions)
Ab8226, by Abcam (4 mentions)
Ab205718, by Abcam (4 mentions)
Ab101562, by Abcam (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab32072, by Abcam (3 mentions)
Ab6721, by Abcam (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ab104836, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab18203, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Ab176316, by Abcam (2 mentions)
Ab151432, by Abcam (2 mentions)
Ab243861, by Abcam (2 mentions)
Ab282176, by Abcam (2 mentions)
Ab32391, by Abcam (2 mentions)
Ab75814, by Abcam (2 mentions)
24 protocols using ab137852
1
Immunoblotting Analysis of Protein Expression
2
Evaluating Protein Expression in Breast Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were isolated from breast cancer tissues and cells using RIPA lysis buffer (Sigma-Aldrich; Merck KGaA). The protein concentration was measured with BCA Protein Assay kit (CoWin Biosciences), and the protein (60 µg) was separated on a 10% SDS-PAGE gel. After transferring separated proteins onto polyvinylidene difluoride membrane (EMD Millipore), membranes were blocked with 5% non-fat milk for 2 h at 4°C and then incubated with the following primary antibodies: Rabbit anti-GAPDH (1:1,000; ab8245; Abcam), anti-NUAK2 (1:1,000; ab224079; Abcam), anti-glucose transporter 1 (GLUT1; 1:1,000; ab115730; Abcam), anti-pyruvate kinase muscle isozyme M2 (PKM2; 1:1,000; ab137852; Abcam) and anti-lactate dehydrogenase A (LDHA; 1:1,000; ab101562; Abcam). Following overnight incubation at 4°C, membranes were washed three times and incubated with peroxidase-labeled secondary antibody (anti-rabbit IgG, 1:2,000; ab6721; Abcam) at room temperature for 2 h. Protein bands were visualized using enhanced chemiluminescence system (ECL; Thermo Fisher Scientific, Inc.). Image Lab Software (version 1.8.0; Bio-Rad Laboratories, Inc.) was used for quantification.
3
Protein Expression Analysis in OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OS cells were collected and then washed using phosphate-buffered saline buffer (PBS; Sangon Biotech) for three times. Cell lysates were prepared using whole cell lysis buffer (Invitrogen). Protein samples were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and dry-transferred onto polyvinylidene difluoride (PVDF) membrane (150 V/2 h; Bio-Rad, Hercules, CA, USA). The non-specific sites in the membrane were sealed using 5% non-fat milk. The diluted primary antibodies of anti-CEP55 (ab170414; Abcam, Cambridge, MA, USA) at the dilution of 1:10,000, anti-hexokinase 2 (HK2; ab227198; Abcam) at the dilution of 1:20,000, anti-pyruvate kinase M 2 (PKM2; ab137852; Abcam) at the dilution of 1:3000 and anti-β-actin (ab8226; Abcam) at the dilution of 1:20,000 were incubated with the membrane overnight. Afterwards, the membrane was labeled with diluted horseradish peroxidase (HRP)-labeled secondary antibody (Abcam) at the dilution of 1:5000. Immunoreactive protein bands were assessed by the enhanced chemiluminescence (ECL) kit (Pierce, Waltham, MA, USA). The quantification of protein bands was performed using Image Lab analysis software (Bio-Rad).
4
Western Blot Analysis of PKM2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein samples were diluted with 5× loading buffer and then subjected to electrophoretic concentration for 30 min and separation for 70 min. The reaction was terminated in PBS blocking buffer which contains 5% (w/v) skim milk powder for incubation at room temperature for 30 min. After that, the proteins were incubated overnight with primary antibody of PKM2 (1:500, ab137852, Abcam). After PBS washing, the membranes were incubated with secondary antibody for 1 h at room temperature. The blots were photographed under a Gel Doc XR Imaging System (BIO‐RAD).
5
Protein Expression Analysis Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The loading protein samples were prepared as previously described.23 After 10% separation gel treatment, the protein was electro‐transferred on polyvinylidene difluoride membranes (Roche). After being incubated with primary anti‐proliferating cell nuclear antigen (anti‐PCNA) antibody (ab29; Abcam, 1:1000), anti‐MMP9 antibody (ab76003; Abcam, 1:1500), anti‐hexokinase‐2 (anti‐HK2) antibody (ab209847; Abcam, 1:1500), anti‐M2 isoform of pyruvate kinase (anti‐PKM2) antibody (ab137852; Abcam, 1:1000), anti‐ICMT (NBP1‐69579; Bio‐Techne, 1:1500 dilution), or loading control anti‐β‐actin (ab8226; Abcam, 1:3000), the membranes were mixed with secondary antibody (ab205718; Abcam, 1:5000). Protein signals were activated and detected ECL kit.
6
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The radioimmunoprecipitation assay (RIPA) buffer was used to extract total protein, supplemented with 1% protease inhibitors (P8340, Sigma-Aldrich) and phosphatase inhibitors (P5726, Sigma-Aldrich). The bicinchoninic acid (BCA) assay was used to measure protein concentration. Western blot analysis was performed, as previously described (12 (link)). THBS2 (sc-136238, Santa Cruz Biotechnology), TLR4 (ab30667, Abcam), Ki67 (Proteintech Group, Inc.), HIF-1α (ab2185, Abcam), GLUT1 (ab115730, Abcam), HK2 (ab104836, Abcam), ALDOA (ab150396, Abcam), PKM2 (ab137852, Abcam), and LDHA (ab101562, Abcam) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (H+L) and HRP-conjugated AffiniPure goat anti-mouse IgG (H+L) were obtained from Proteintech Group, Inc. (Jackson).
7
Protein Expression Analysis in PC12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For determining the protein expression of PKM2, Brg1 and STAT3, Western blot was used. Whole cell extracts, including cytosolic protein extracts and nuclear protein fraction, were obtained from PC12 cells using the lysis buffer as described previously.29 (link) An equal amount of total protein samples (40 μg) was loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to PVDF membranes (Millipore, Bedford, MA, USA). Nonfat milk (5%) was used to block the reaction for 1 h at room temperature, and then the primary antibodies were added to the members overnight at 4 °C. Following antibodies were used in this study: PKM2 (ab137852, 1:1000, Abcam, Cambridge, MA, USA), Brg1 (ab4081, 1:1000, Abcam), p-STAT3 (ab76315, 1:5000, Abcam) and STAT3 (ab68153, 1:1000, Abcam). Horseradish peroxidase-conjugated secondary antibody (ab6721, 1:2000, Abcam) was incubated with the members for 1 h at room temperature. The ECL reagent was used to detect the immunoblots.
8
Immunoprecipitation and Immunoblotting of PKM2 and Ubiquitin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MG63 cells that transfected with oeTRIM58 were lysed in 1% SDS-containing radio immunoprecipitation assay (RIPA) buffer by sonication on ice. Then, lysates were treated with Protein A/G Plus-Agarose (sc-2003, Santa Cruz, USA) for 1 h. After that, each sample was incubated with the IgG (30000-0-AP, Proteintech, USA) overnight at 4°C. Then, the nuclear pellet was collected by centrifugation at 3000 rpm for 5 min at 4°C and subsequently washed four times by Protein A/G Plus-Agarose beads. The purified proteins were separated by gradient SDS-PAGE. Anti-PKM2 (ab137852, Abcam, UK) or anti-ubiquitin antibody (ab7780, Abcam, UK) was used for immunoblotting.
9
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were isolated using radioimmunoprecipitation assay (RIPA; Solarbio, Beijing, China) buffer with proteinase inhibitor. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then blotted onto the polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After blocking with 5% nonfat milk, the membrane was incubated with the following primary antibodies overnight: anti-E-Cadherin (ab1416; Abcam, Cambridge, MA, USA), anti-Vimentin (ab92547; Abcam), anti-N-Cadherin (ab18203; Abcam), anti-HIF-1α (ab16066; Abcam), anti-glucose transporter 1 (anti-GLUT1; ab40084; Abcam), anti-phosphoglycerate kinase 1 (anti-PGK1; ab199438; Abcam), anti-pyruvate kinase M2 (anti-PKM2; ab137852; Abcam), and GAPDH (ab181602, Abcam) were used as the internal references for immunoblot. Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) were used to incubate with the membrane the next day. Protein signals were visualized with the enhanced chemiluminescence detection kit (Millipore, Billarica, MA, USA).
10
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 collected, lysed, and denatured. The Bradford method was performed to evaluate the amount of proteins. Forty micrograms of protein was separated using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. The protein was transferred to a polyvinylidene fluoride membrane using the electrotransfer method after being blocked with skimmed milk for 2 h. Subsequently, the blots were visualized with primary antibody incubated overnight at 4°C, followed by goat anti-rabbit secondary antibody incubation for 2 h. The blots were visualized with the ECL Fluorescence Detection Kit. Photographs were taken and the ImageJ software was used to calculate the gray value of each band. The primary antibodies used were as follows: FOXP3 (ab20034, 1:1,000, Abcam), PKM2 (ab137852, 1:1,000, Abcam), and GAPDH (ab8245, 1:1,000, Abcam).
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!