Whole-cell extracts (WCE) were prepared by lysing cells in two volumes of Lysis buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L HEPES, 1% v/v Triton-X100, 0.1% v/v SDS). Thirty to 60 μg of WCE was resolved on 4%–12% Bolt Gels (Thermo Fisher Scientific). Protein was transferred to nitrocellulose membrane (Thermo Fisher Scientific) and blotted for SF3B1 (Bethyl Laboratories: A300–996A; RRID:AB_805834)), Flag M2 (Sigma-Aldrich: F3165; RRID:AB_439685), mRFP (Origene:TA180093; RRID:AB_2622287) and γ-tubulin (GTU-88:Sigma Aldrich; RRID:AB_532292), ATM (2C1:Abcam; RRID:AB_368161), BRCA2 (OP95:Millipore; RRID: AB_2067762), ATR (N19:SCBT; RRID:AB_630893), Mre11 (4895:Cell Signaling Technology; RRID:AB_2145100), BRCA1 (D-9:SCBT; RRID:AB_626761), RAD51 (SCBT:3C10/sc-53428; RRID:AB_630180), γH2AX (Millipore:JBW301/05–636; RRID:AB_309864), GAPDH (Sigma: HPA040067; RRID:AB_10965903), vinculin (Abcam:ab219649; RRID: AB_2819348), β-actin (SCBT:C4; RRID: AB_2714189), and GFP (D5.1:Cell Signaling Technology; RRID:AB_1196615).
Ab219649
Manufactured by Abcam
Sourced in China
Ab219649 is a laboratory reagent from Abcam. It is a polyclonal antibody that recognizes an epitope within the KIF5B protein. The antibody is suitable for use in various immunoassays, such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
F3165, by OriGene (2 mentions)
Bolt gel, by Thermo Fisher Scientific (2 mentions)
Gtu 88, by Merck Group (2 mentions)
Ta180093, by OriGene (2 mentions)
Jbw301 05 636, by Merck Group (2 mentions)
Hpa040067, by Abcam (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab273012, by Abcam (1 mentions)
Chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
Bca protein detection kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Anti mouse a4416, by Merck Group (1 mentions)
β actin actb, by Merck Group (1 mentions)
Mre11, by Cell Signaling Technology (1 mentions)
γ tubulin, by Merck Group (1 mentions)
Ab187149, by Abcam (1 mentions)
Anti pkm2 antibody, by Cell Signaling Technology (1 mentions)
9 protocols using ab219649
1
Whole-Cell Extraction Western Blot
2
Western Blot Analysis of Cellular Markers
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The western blot experiment was conducted in accordance with standard procedures. The primary antibodies used for this research were as follows: anti-ZSCAN18 (TA505326, OriGene), anti-VINCULIN (ab219649, Abcam), anti-p62/SQSTM1 (#16177S, CST), anti-LC3A/B (#12741S, CST) and anti-TP53INP2 (ab273012, Abcam).
3
Western Blot Analysis of Protein Targets
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Total protein was subjected to SDS-PAGE and transferred to the PVDF membrane (IPVH00010, Millipore). The antibody against CTSW (ab191083), KLF5 (21017-1-AP), or vinculin (ab219649) was obtained from Abcam or Proteintech. The membrane was incubated with the primary antibody and visualized using the Chemiluminescent Substrate Kit (34580, Thermo Fisher).
4
Muscle Tissue Protein Extraction and Analysis
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Total protein of muscle tissues was extracted using RIPA lysate (Beyotime, Shanghai, China). BCA protein detection kits (Thermo Fisher Scientific, Waltham, MA, United States ) were used to determine the protein concentrations of each group. The same amounts of protein were separated using 8% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Thereafter, PVDF membranes were sealed with 5% skim milk powder in 0.1% Tween’s Tris-buffer saline for 2 h, and subsequently incubated with specific primary antibodies against IL18R1 (DF7059, Affinity, China), vinculin (ab219649, Abcam, China) overnight at 4°C. Thereafter, the anti-rabbit IgG enzyme-conjugated secondary antibody (Proteintech, Wuhan, China) was incubated at room temperature for 1 h. Enhanced chemiluminescence (Bio-RAD, Hercules, CA, United States ) was used to detect western blot results.
5
Antibody Dilution Protocol for Protein Analysis
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Antibodies from Abcam were diluted as follows: recombinant anti-PIP5K1C (ab109192) 1:1000, PIP5K1B (ab154818) 1:1000, CHUK/IKK alpha 1:1000 and VCL/vinculin (ab219649) 1:10,000. Antibodies from Cell Signaling Technology were diluted as follows: PIP5K1A (9693) 1:1000 SQSTM1 (8025) 1:5000, LC3A/B (12,741) 1:5,000, p-RPS6 S240/244 (5364) 1:20,000, RPS6 (2217) 1:20,000, and HRP-conjugated secondary anti-rabbit (7074) 1:10,000. Antibodies from Sigma-Aldrich were PIP4K2C (SAB1407977) 1:10,000, ACTB/β-actin (A5441) 1:20,000, and anti-mouse (A4416) 1:10,000. Other antibodies were PIKFYVE (Millipore, MABS522) 1:1000
6
Whole Cell Proteome Analysis
7
Western Blot Analysis of Protein Expression
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Western blotting was performed on extracted protein samples using anti-SMYD3 (Abcam, ab187149, 1:1000 dilution), and anti-vinculin (Abcam, ab219649, 1:1000 dilution) antibodies as well as anti-PKM2 antibody (Cell Signaling Technology, #4053, 1:3000 dilution). Vinculin was used as the loading control. Briefly, harvested cells were washed with cold PBS and then lysed in RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein was separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, MA, USA). The membranes were incubated with antibodies overnight at 4 °C. After incubation, the membranes were washed with TBST (1*TBS containing 0.1% Tween-20), followed by incubation with HRP-conjugated secondary antibody for 1 h at room temperature. Washed again with TBST, the immunoreactive bands were visualized with Bio-rad Image analysis systems (Bio-Rad, Hercules, CA, USA).One loading control was performed for the proteins on the same membrane. Full length western blots were provided in the Supplementary materials .
8
KIF15 Protein Quantification by Western Blot
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Cells were lysed in protein lysis buffer, vortexed on ice for 10 min (vortexing for 30 s and incubation on ice for 15 s) and centrifuged at 4°C at 12,000 rpm for 5 min. The supernatant (total 20 μg of protein) was mixed with loading buffer and boiled for 5 min. Subsequently, 5% stacking and 10% separation gel was prepared for electrophoresis. After electrophoresis at 120 V for 60 min and 350 mA for 60 min, the proteins were transferred on the PVDF membranes. Then, the membranes were incubated with primary anti-KIF15 (#ab272615, Abcam, 1:1000) and anti-vinculin antibodies (#ab219649, Abcam, 1:500) and with secondary antibodies. Relative grayscale intensity of the bands was analyzed using the ImageJ software. Tubulin was used as the internal standard.
9
Western Blot Analysis of Cell Signaling Proteins
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The cells were collected and lysed with immunoprecipitation assay buffer (NP40 buffer) added with protease inhibitor cocktail (Roche Applied Science). Lysates were fractionated by 10% SDS-PAGE and subsequently transferred onto nitrocellulose membranes (Hybond C; GE Healthcare Life Sciences). The membranes were blocked with 5% non-fat milk for obstructed non-speci c binding sites at room temperature for 1 h and then incubated with the following antibodies overnight at 4˚C: Anti-NAT10 (ab194297; dilution, 1:1000; Abcam), CDK7 (ab256787; dilution, 1:1000; Abcam), Vinculin (ab219649; dilution, 1:1,000, Abcam), CDK1 (ab133327; dilution, 1:1,000, Abcam), CDK2 (ab32147; dilution, 1:1,000, Abcam) Phospho-CDK1 (ab201008; dilution, 1:1,000, Abcam), Phospho-CDK2 (#2561; dilution, 1:1,000, Cell Signaling Technology). Membranes were then washed three times and then incubated with HRPconjugated goat anti-rabbit (#7074; dilution, 1:500) or goat anti-mouse (#7076; dilution, 1:500) antibodies (both from Cell Signaling Technology). The bands were visualized by enhanced chemiluminescence reagents (cat. no. WP20005; Thermo Fisher Scienti c, Inc.).
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