The protein level of TRIM16 was detected using Western blot, according to the experimental procedures recorded in the previous publications [29 (link)]. Briefly, transfected cells were lysed in Radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitor (Thermo Fisher Scientific) to extract total protein. The protein quantification was measured by the BCATM Protein Assay Kit (Pierce, Appleton, WI, USA). Then the equivalent protein (50 µg) was added to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain the target protein. Next, the target proteins in the gel were transferred onto polyvinylidene difluoride (PVDF) membranes and incubated with primary antibodies against TRIM16 (ab72129, 1:2000, Abcam, Cambridge, MA, USA), hexokinase 2 (HK2, ab227198, 1:5000, Abcam), B-cell lymphoma protein 2 (Bcl-2)-associated X (BAX, ab32503, 1:2000, Abcam), Matrix metalloproteinase 2 (MMP2, ab92536, 1:1000, Abcam), Proliferating cell nuclear antigen (PCNA, ab18197, 1:1000, Abcam), and β-actin at 4°C overnight. The membranes were then incubated with second antibodies (Horseradish peroxidase-conjugated IgG antibody). Finally, the Western blot intensities were detected using LAS 4000 Image Reader (Fujifilm, Tokyo, Japan)
Ab227198
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab227198 is a recombinant monoclonal antibody. It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (8 mentions)
Ab9485, by Abcam (8 mentions)
Ab8227, by Abcam (7 mentions)
Ab125683, by Abcam (5 mentions)
Ab18197, by Abcam (4 mentions)
Ab8226, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab15148, by Abcam (3 mentions)
Ab37168, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Ripa buffer, by Solarbio (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab32503, by Abcam (2 mentions)
Pvdf membrane, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ab97051, by Abcam (2 mentions)
Ecl kit, by Solarbio (2 mentions)
Ab115730, by Abcam (2 mentions)
30 protocols using ab227198
1
TRIM16 Protein Quantification via Western Blot
2
Protein Expression Analysis in OS Cells
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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).
3
Western Blot for Protein Expression
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At 48 h post-transfection, OS tissues and cells were collected and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing phenylmethanesulfonyl fluoride (PMSF; Beyotime) to obtain total protein. The protein concentrations of the total cellular lysates were quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime). An equal amount (40 µg) of protein was resolved by 810% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After that, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (0.2 μm, Beyotime). Next, all membranes were blocked using the 5% non-fat milk and then probed with the following primary antibodies: antibodies against hexokinase II (HK2) (1:5000, ab227198, 102 kDa), E-cadherin (1:500, ab15148, Abcam), Vimentin (1:2500, ab92547, Abcam), β-actin (1:2500, ab8227, Abcam), MAPK7 (1:200, ab92547, Abcam) and GAPDH (1:2000, ab37168, Abcam). Subsequently, all membranes were maintained in HRP-conjugated anti-rabbit/mouse IgG (Sangon Biotech). Finally, all protein bands were observed using the enhanced chemiluminescence (ECL) system (GEHeathcare, Waukesha, WI, USA). The protein abundances were normalized with GAPDH and β-actin, and ImageJ software was applied to evaluate the density.
4
Western Blot Analysis of Protein Levels
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The proteins were extracted from cells or tissues using RIPA buffer (Beyotime) with protease inhibitors. The BCA Kit was applied to the detection of protein concentrations. Then equal amounts (20 μg) of proteins denatured by boiled water bath were separated by SDS-PAGE and then transferred onto 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in TBST with 5% non-fat milk for 1 h and then incubated with rabbit anti-human primary antibodies against HK2 (ab227198, 1:5000 dilution, Abcam, Cambridge, MA, USA), RPN2(ab244399, 1:2000 dilution, Abcam) or β-actin(ab227387, 1:10000 dilution, Abcam) as a loading control at 4 °C overnight and goat anti-rabbit secondary antibody (ab97051, 1:10000 dilution, Abcam) at room temperature for 2 h. The signals were developed using ECL Kit (Beyotime) and the relative protein levels of HK2 and RPN2 were normalized to the control group.
5
Western Blot Analysis of Metabolic Enzymes
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Protein samples were acquired using radioimmunoprecipitation assay (RIPA) buffer (Beyotime) and then were loaded onto separating gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After sealing with 5% (w/v) nonfat milk for 1 h, the membrane was incubated with primary antibodies (Abcam, Cambridge, MA, USA) containing anti-hexokinase 2 (anti-HK2, ab227198), anti-lactate dehydrogenase A (anti-LDHA, ab125683), anti-PDE4D (ab249652), and anti-β-actin (ab8227). The membrane was labeled with appropriate secondary antibody (Abcam), and protein bands were visualized using the enhanced chemiluminescent substrate (Amersham Biosciences, Piscataway, NJ, USA).
6
Western Blot Analysis of Apoptosis and Metabolism Markers
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Western blot was performed according to a previous description.18 (link) In brief, proteins were extracted from samples by using the total protein extraction kit (Solarbio) and then the proteins were separated and incubated with the primary antibodies and the secondary antibody. The antibodies used in this research: anti-B-cell lymphoma-2 (Bcl-2) (1:2000, ab196495, Abcam), anti-BCL2-Associated X (Bax) (1:1500, ab199677, Abcam), anti-hexokinase 2 (HK2) (1:5000, ab227198, Abcam), anti-MAGT1 (1:3000, ab90478, Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam) and goat anti-rabbit IgG H&L (HRP) (1:3000, ab205718, Abcam). After the treatment with ECL kit (Solarbio), the protein band was analyzed using the ChemiDoc™ MP Imaging System (Bio-Rad).
7
Protein Extraction and Western Blot Analysis
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Extraction of total protein was executed using the RIPA lysis buffer (Beyotime), and western blotting was executed as previously described.25 Antibodies used in the study were as follows: matrix metalloproteinase 2 (MMP2) (ab97779, 1:2000, Abcam), matrix metalloproteinase 9 (MMP9) (ab228402, 1:1000, Abcam), hexokinase 2 (HK2) (ab227198, 1:10000, Abcam), lactate dehydrogenase A (LDHA) (ab125683, 1:1000, Abcam), and NUCKS1 (PA5‐26535, 1:1000, Invitrogen), anti‐β‐actin (ab115777, 1:200, Abcam), and goat anti‐rabbit IgG (ab6721, 1:10000, Abcam). Protein bands were visualized with enhanced chemiluminescence solution (Beyotime).
8
Protein Expression Analysis by Western Blotting
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Protein lysates were harvested using RIPA lysis buffer with freshly added protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes (Millipore, Bedford, USA), which were then blocked with 5% skim milk and incubated with RNF7 (ab181986; 1:5000), SOCS1 (ab62584; 1:600), HK2 (ab227198; 1:8000), STAT3 (ab119352; 1:2000), p-STAT3 (ab76315; 1:5000; all from Abcam), GLUT1 (orb157188; 1:2000; Biorbyt, St Louis, MO, USA), cleaved PARP1 (#5625; 1:1000), cleaved caspase-3 (#9661; 1:1000), and GAPDH (#5174; 1:2000; all from Cell Signaling Technology, Danvers, MA, USA) primary antibodies and HRP-conjugated secondary antibodies (A0208, A0181; 1:1000; Beyotime). Protein bands were visualized using an enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA). Protein levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
9
Western Blot Analysis of EMT Markers
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The analysis of Western blot was carried out as previously described.18 (link) Protein extracts were separated on an 8–10% SDS polyacrylamide gel electrophoresis, followed by the transferring to a nitrocellulose membrane (Millipore). The primary antibodies used in the experiments were as follows: anti-E-Cadherin (ab1416), anti-Vimentin (ab8978), anti-N-Cadherin (ab76057), anti-HK2 (ab227198) and anti-GAPDH (ab181602) (all from Abcam, Cambridge, UK).
10
Western Blot Analysis of Cellular Proteins
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Total cellular and tissue proteins were extracted with Radio-Immunoprecipitation assay (RIPA) cell lysis buffer, followed by estimation of protein concentration with a bicinchoninic acid kit. A corresponding volume of proteins were added into and mixed evenly with loading buffer, after which they were denatured by 3-min heating in a boiling-water bath. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was utilized to transfer proteins to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Subsequent to 60-min blocking in 5% skim milk powder, the membrane was probed overnight in a shaker at 4°C with primary antibodies (Abcam, Cambridge, UK) against SMYD2 (ab259973, 1:1000), GAPDH (1:10000, ab9485), APOC1 (1:1000, ab231570), Hexokinase 2 (HK2, 1:5000, ab227198), LDHA (1:5000, ab52488), and Glucose Transporter Type 1 (GLUT1, 1: 5000, ab115730). The next day, the membrane was washed by Tris-buffered saline with Tween 20 (TBST) and then incubated for 2 h at room temperature with goat anti-rabbit Immunoglobulin G (IgG, 1:5000, ComWin, Beijing, China). Subsequent to 3 washes with TBST, electrogenerated chemiluminescence (ECL) liquid was utilized to capture images in an ECL imaging analysis system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
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