Proteins were extracted from bone marrow tissue or BMSCs with Radio Immunoprecipitation Assay (RIPA) lysis buffer containing protease inhibitors on ice. Equal quantities of protein were segregated using 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with non-fat dry milk, the membranes were incubated with specific primary antibodies (Abcam, MA, USA). The membranes were then incubated with HRP-conjugated secondary antibody. β-actin was used as an internal reference protein.
Specific primary antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Specific primary antibodies are laboratory reagents used to identify and detect target proteins or antigens in biological samples. They are designed to bind to a specific epitope or region of a target molecule with high affinity and selectivity.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (8 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Protein assay system, by Bio-Rad (2 mentions)
Ecl detection reagent, by Cytiva (2 mentions)
Bca kit, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Roche (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca kit, by Bio-Rad (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Il 18, by Abcam (1 mentions)
Gsdmd, by Abcam (1 mentions)
Hmgb1, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Hrp substrate, by Merck Group (1 mentions)
Protein extraction kit, by Keygene (1 mentions)
Protease inhibitor cocktail, by Transgene (1 mentions)
Mammalian total protein extraction kit, by Transgene (1 mentions)
40 protocols using specific primary antibody
1
Western Blot Analysis of Bone Marrow
2
Western Blot Analysis of Protein Expression
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Cells were treated with RIPA buffer (Beyotime, Shanghai, China) to extract total proteins. After quantitation using a BCA kit (Bio-Rad, Hercules, CA, USA) and separation by SDS-PAGE, protein samples were electroblotted onto nitrocellulose membranes (Millipore, Billerica, MA, USA). After sealed with 5% fat-free milk for 1 h, the membranes were incubated overnight with the specific primary antibodies (Abcam, Cambridge, UK) against SIAH2, HIF-1α, VE-cadherin, MMP-9, EphA2, E-cadherin, N-cadherin, β-actin, and Snail at 4°C, followed by 1 h of incubation with the HRP-linked secondary antibodies at room temperature. The protein bands were finally visualized and analyzed by ECL detection system and ImageJ software.
3
Apoptosis Signaling Pathway Evaluation
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Western blot assay was used to evaluate the levels of apoptosis-related proteins (Bax, Bcl-2, pro-caspase3, and cleaved caspase3) and signaling pathway-related factors (p-p38, p38, p-p65, and p65). The neuronal cell total proteins were separated and quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (Sigma, USA). Then, proteins (30 μg/lane) were isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto a polyvinylidene fluoride (PVDF) membrane. After incubating in 5% skim milk for 2 h at room temperature, the membranes were cultured at 4°C in specific primary antibodies (Abcam) at 1: 1000 dilution. GAPDH was used as internal control overnight. Subsequently, the membranes were cultured with secondary antibody (Abcam; 1: 2000 dilution) for 2 h. The protein bands were detected by enhanced chemiluminescence (ECL) detection system reagents (GE Healthcare Life Sciences, UK) following the manufacturer’s instructions. Proteins were quantified using ImageJ software (version 1.38X; National Institutes of Health).
4
Pyroptosis Protein Expression Analysis
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The expression of proteins associated with pyroptosis were measured by Western blot analysis with specific primary antibodies against NLRP3 (1:800), Caspase 1 (1:1000), GSDMD (1:500), IL-18 (1:1000), IL-1β (1:1000) and HMGB1 (1:500) (all from Abcam, UK).
5
Western Blot Analysis of Treg Proteins
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In the Western blot analysis, proteins were extracted from Tregs using a protein extraction kit (KeyGene, NED). The extracted proteins were added to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by migration with 120 V electric tension, and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skimmed milk powder in Tris-buffered saline containing Tween 20 (TBST), and incubated with specific primary antibodies (Abcam, USA) at 4°C overnight. The following day, the membranes were washed for 15 min three times in TBST, and incubated with secondary antibodies (Abcam, USA) for 2 h at room temperature. HRP Substrate (Millipore, USA) was used to visualize the protein bands, and the band intensities were quantified using Image Pro plus software (Mediacy, USA). For more detailed steps, please refer to the antibody specification and protocol description.
6
Western Blot Analysis of Protein Expression
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Cultured cells were lysed in lysis buffer containing protease inhibitor. Protein concentration was determined using a Bio-Rad protein assay system (Bio-Rad). Equivalent amounts of proteins were separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Bio-Rad). After being blocked in Tris buffered saline (TBS) containing 5% nonfat milk, the membranes were incubated with specific primary antibodies (Abcam) at 4°C for 12 hours and then with horseradish peroxidase conjugated anti-mouse antibody for 2 hours at room temperature. ECL detection reagent (Amersham LifeScience) was used to demonstrate the results.
7
Evaluating Protein Expression Profiles
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Using a Mammalian Total Protein Extraction Kit and a protease inhibitor cocktail, the cells' total proteins were extracted (Transgen, Beijing, China). In a nutshell, 10% SDS–polyacrylamide gel electrophoresis was used to separate the proteins. Following that, the proteins were transferred to PVDF membranes. Specific primary antibodies (Abcam, UK) were incubated with the PVDF membranes for an extended period of time at 4 °C in order to identify PD-L1, P65, p-P65, and GAPDH. The membranes were then exposed to peroxidase-conjugated secondary antibodies for two hours at room temperature. Finally, an enhanced chemiluminescence assay was used to see the Western blotting bands (ECL; Thermo Fisher, USA).
8
Western Blot Analysis of Proteins
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Proteins lysates obtained in RIPA lysis buffer were subjected to 10% SDS‐PAGE and transferred onto the PVDF membrane. Membranes were sealed with 5% skimmed milk and incubated with specific primary antibodies (Abcam) overnight at 4°C. Followed by washing in TBST, secondary antibodies conjugated with HRP were used. Proteins were visualized using a detection system of enhanced chemiluminescence (ECL). GAPDH served as the internal reference.
9
Protein Expression Analysis in A549 Cells and Tumor Tissues
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Using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), total proteins were extracted from A549 cells and tumor tissues. Protein concentration was determined by a BCA kit (Beyotime Institute of Biotechnology). The proteins (25 µg) were separated in 10% SDS-PAGE gels and then transferred to PVDF membranes, which were blocked with 5% non-fat dry milk for 1 h at room temperature. After incubation with specific primary antibodies (all purchased from Abcam) at 4°C overnight, the membranes were washed thrice with 1X 0.05% TBST for 10 min and incubated with goat-anti-rabbit IgG H&L secondary antibodies (1:2,000; cat no. ab6721; Abcam) at room temperature for 1 h. Protein bands were visualized by an ECL reagent (Applygen Technologies, Inc.). The primary antibodies were as follows: NPM1 (1:400; cat no. ab183340), phosphorylated (p)-EGFR (1:1,000; cat no. ab40815), EGFR (1:5,000; cat no. ab52894), p-MEK (1:2,500; cat no. ab96379), MEK (1:2,500; cat no. ab32091), p-ERK (1:1,000; cat no. ab201015), ERK (1:10,000; cat no. ab184699), GAPDH (1:2,500; cat no. ab9485). The protein bands were visualized using an ECL kit (Applygen Technologies, Inc.), and the relative protein levels were quantified using ImageJ software 1.8.0 (National Institutes of Health).
10
ADSC Osteogenesis Marker Expression
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Immunocytochemistry was performed according to a previously reported study36 (link). In brief, transduced ADSCs were washed with phosphate saline (PBS, Invitrogen) and fixed in 4% paraformaldehyde (Sigma), then permeabilized in Triton X-100 (1%, Sigma) for 10 minutes. Specific primary antibodies targeting Runx2 and Osterix (1:200, all from Abcam) were incubated overnight before using species-specific fluorescence-conjugated secondary antibody (1:800, Abcam). Nuclei were stained with DAPI (Sigma) before imaged on a confocal microscope (Leica microsystems, Heidelberg, Germany). Positive cell ratio was evaluated by dividing the number of Runx2- or Osterix-positive ADSCs by the number of DAPI-stained ADSCs, respectively.
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