sEV protein content was measured by BCA, and 10 μg aliquots were used for Western blot analysis. sEVs were lysed in Laemmli buffer at 96°C for 5 minutes to denature proteins, and protein extracts were resolved by SDS-PAGE. Antibodies were tested against albumin (sc-271605, Santa Cruz Biotechnology), apolipoprotein B (ab139401, Abcam), TSG101 (ab125011, Abcam), CD9 (ab58989, Abcam), and ALIX (ab88743, Abcam). Antibodies were chosen according to MISEV 2023 guidelines (21 (link)). Immunostaining against PAX8 (PA 0300, Biopat), a well-known ovarian carcinoma marker, was also incorporated in the Western blot characterization panel. The biological validation used antibodies against STX5 (HPA001358, Sigma-Aldrich) and S100A4 (ab93283, Abcam). Clarity ECL Western Blotting Substrate (Bio-Rad) was used to visualize the bands and developed on the ChemiDoc imaging system (Bio-Rad). The intensity of the immunoreactive bands was quantified by densitometry using ImageJ (NIH).
Ab88743
Manufactured by Abcam
Sourced in United States
Ab88743 is a mouse monoclonal antibody that recognizes the human EGF receptor. The antibody can be used for various applications, including immunohistochemistry, immunocytochemistry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Ab125011, by Abcam (2 mentions)
Ab139401, by Abcam (1 mentions)
Ab58989, by Abcam (1 mentions)
Ab93283, by Abcam (1 mentions)
Sc 271605, by Santa Cruz Biotechnology (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ab223052, by Abcam (1 mentions)
Ab126729, by Abcam (1 mentions)
Ab169532, by Abcam (1 mentions)
Rabbit anti β3 tubulin, by Cell Signaling Technology (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Ab216130, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Ab236630, by Abcam (1 mentions)
Ab227198, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
6 protocols using ab88743
1
Characterization of Extracellular Vesicles
2
Protein Expression Analysis by Western Blotting
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Total protein extraction was performed using a radioimmunoprecipitation assay solution. Protein concentration was measured using the BCA assay, and an equal quantity of each sample (100 μg) was used for Western blotting. Following SDS-PAGE, the proteins were transferred onto 0.45 µm polyvinylidene difluoride membranes (IPVH00010, Millipore, USA). Membranes were blocked with 5% (w/v) non-fat dry milk in Tris base saline Tween 20 (TBST; 50 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween-20 (v/v)) for 1.5 hours, and then, washed thrice for 10 minutes in TBST, followed by incubation at 4 °C overnight with mouse anti-Alix (ab88743, Abcam), rabbit anti-CD9 (ab223052, Abcam), rabbit anti-Netrin1 (ab126729, Abcam), mouse anti-NF (#2836, Cell Signaling Technology), rabbit anti-microtubule-associated protein (MAP2) (#4542, Cell Signaling Technology), rabbit anti-β3-tubulin (#5568, Cell Signaling Technology), rabbit anti-Phox2b (25276-1-AP, Proteintech Group Inc, USA), rabbit anti-Hand2 (abs117611, Absin Bioscience Inc., China), GAPDH (10494-1-AP, Proteintech Group Inc), and Lamin A/C (ab169532, Abcam). Next, the membranes were washed thrice in TBST and incubated with a secondary antibody at room temperature for 2 hours. Following washing with TBST, the reaction was detected using chemiluminescence and the Western Bright ECL detection kit (WBKLS00100, USA).
3
Evaluating EV Protein Profiles via SDS-PAGE
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hBMSCs‐derived EVs were resuspended in pre‐cooled lysis buffer solution containing protease inhibitor cocktail tablets (Roche Life Science, Basel, Switzerland). The lysate was dissolved in 3 × Laemmli's buffer solution, boiled for 5 minutes, analysed by 12% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto the nitrocellulose membrane. The membrane was then blocked in 0.05% Tween supplemented with 5% skimmed milk and PBS (pH = 7.4), probed with rabbit antibodies (Abcam) to ALIX (ab88743), CD63 (ab216130) and glucose‐regulated protein 94 (GRP94) (ab13509) and re‐probed with diluted horseradish peroxidase‐conjugated secondary antibody of goat anti‐rabbit IgG (ab6721, 1:10000, Abcam). The protein signal was detected by enhanced chemiluminescence (ECL) reagents (170‐8280, Bio‐Rad Laboratories Inc, Hercules, CA, USA), with Ponceau red serving as the internal reference.
4
RNA and Protein Analysis of Tumor Samples
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Extraction of total RNA from indicated tumor tissues or cell lines was done using TRIzol reagent (Takara). Employing 1 μg of total RNA and PrimeScript RT Reagent Kit (Takara), the first-strand cDNA was synthesized. The levels of miRNA were determined by qRT-PCR with SYBR-Green ΙΙ PCR kit (Takara) and U6 snRNA was used as the reference. LncRNA and mRNA concentrations were measured employing SYBR-Green ΙΙ PCR kit (Takara). β-actin was used as the internal control. Supplementary Table S2 shows the sequences of the primers used.
Extraction of protein was from the HCC cells was done using RIPA buffer, and determined using the BCA Protein Assay Kit (Beyotime Biotechnology). Separation of extracted proteins (40 μg per lane) was done by SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad). The PVDF membranes were blocked using 5% non-fat milk for 1 h and primary antibodies against GLUT1 (Biorbyt, St Louis, MO, USA; orb157188), HK2 (Abcam; ab227198), LDHA (Abcam; ab52488), ALDH1A3 (Proteintech; 29,373–1-AP), β-actin (Abcam; ab8226), Alix (Abcam; ab88743), CD9 (Abcam; ab236630) and TSG101 (Abcam; ab125011) were used. Secondary antibodies were labeled with Peroxidase. The ECL detection system (Thermo) was used for visualization.
Extraction of protein was from the HCC cells was done using RIPA buffer, and determined using the BCA Protein Assay Kit (Beyotime Biotechnology). Separation of extracted proteins (40 μg per lane) was done by SDS-polyacrylamide gel electrophoresis and the separated proteins were transferred to polyvinylidenedifluoride (PVDF) membranes (Bio-Rad). The PVDF membranes were blocked using 5% non-fat milk for 1 h and primary antibodies against GLUT1 (Biorbyt, St Louis, MO, USA; orb157188), HK2 (Abcam; ab227198), LDHA (Abcam; ab52488), ALDH1A3 (Proteintech; 29,373–1-AP), β-actin (Abcam; ab8226), Alix (Abcam; ab88743), CD9 (Abcam; ab236630) and TSG101 (Abcam; ab125011) were used. Secondary antibodies were labeled with Peroxidase. The ECL detection system (Thermo) was used for visualization.
5
Protein Expression Analysis in Myocardial Ischemia
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Total protein was extracted from myocardium and cardiomyocytes in the ischemic border area and quantified by BCA kit (Beyotime, Jiangsu, China), followed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was then transferred onto nitrocellulose membranes and the membranes was incubated with the following rabbit primary antibodies against: TXNIP (1 : 1000, ab188865, Abcam Inc., Cambridge, MA, USA), B-cell lymphoma-2 (Bcl-2) (1 : 1000, ab59348, Abcam), Bcl-2 associated protein X (Bax) (1 : 1000, #14796, Cell Signaling Technology Company, MA, USA), c-Jun (1 : 1000, #9165, Cell Signaling Technology), CD9 (1 : 2000, ab92726, Abcam), GAPDH (1 : 10000, ab108950, Abcam), CD63 (1 : 1000, ab181602, Abcam), Alix (1 : 1000, ab88743, Abcam) and glucose regulated protein 94 (GRP94) (1 : 1000, ab13509, Abcam). Then, horse radish peroxidase (HRP)-conjugated secondary antibody of goat anti-rabbit or goat anti-rat antibody (1 : 10000, ZSGB-BIO, Beijing, China) was added to incubate with the membrane. Enhanced chemiluminescence (Invitrogen) was performed for development.
6
Exosome Protein Characterization by Western Blot
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The exosomes, cells, and tissues collected after treatment were lysed and homogenized with RIPA lysis buffer (Beyotime). After centrifugation at 8,000 g for 10 min, the samples were quantified by BCA method, denatured at 95°C, and loaded at equal amounts onto the SDS-PAGE gels. The proteins were resolved by electrophoresis and then transferred to PVDF membranes (Millipore). Subsequently, the blots were blocked with 5% non-fat milk at room temperature for 1 h and incubated with the primary antibodies including Alix (ab88743, 1:1,000, Abcam), CD63 (ab59479, 1:1,000, Abcam), TSG101 (ab133586, 1:2,000, Abcam), α-SMA (ab32575, 1:3,000, Abcam), CD31 (ab24590, 1:500, Abcam), VE-cadherin (ab166715, 1:1,000, Abcam), VEGFR2 (ab2349, 1:400, Abcam), ELF5 (ab222251, 1:1,000, Abcam), SP1 (ab227383, 1:5,000, Abcam), Lamin A (ab26300, 1:1,000, Abcam) and GAPDH (ab8245, 1:10,000, Abcam) at 4°C overnight. Following incubation with horseradish-peroxidase-conjugated secondary antibodies, the immuno-activities were visualized with the ECL Kit (GE healthcare) and semi-quantified using ImageJ software1 .
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