Samples of protein extracts were combined with an equal volume of sodium dodecyl sulfate sample buffer. Samples were resolved on 9% polyacrylamide gels, and the separated proteins transferred to polyvinylidene difluoride membranes. Membranes were probed with antibodies against uncoupling protein 1 (UCP1, 1:1,000, #sc-6528, Santa Cruz Biotechnology), hormone-sensitive lipase (HSL, 1,000, #4107, Cell Signaling), phospho-HSL (1:1,000, #4139, Cell Signaling), adipose triglyceride lipase (ATGL, 1:1,000, #2439, Cell Signaling), fatty acid synthase (FAS, 1:2,500, #610962, BD Transduction Laboratories), or β-actin (60008-I-Ig, 1:50,000, Proteintech) followed by a secondary anti-rabbit antibody (1:10,000, #7074, Cell Signaling), anti-mouse antibody (1:10,000, #7076, Cell Signaling), or anti-goat antibody (1:1,000, #sc-2354, Santa Cruz Biotechnology). The protein level was visualized with an enhanced chemiluminescent detection kit (GE healthcare) and quantified by using ImageJ software.
60008 1 ig
Manufactured by Proteintech
The 60008-I-Ig is a protein A affinity resin designed for the purification of immunoglobulins (Ig). It is a ready-to-use agarose-based resin that binds to the Fc region of various Ig isotypes, enabling the efficient capture and isolation of Ig from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescent detection kit, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
A8592, by Merck Group (1 mentions)
Hrp 60004, by Proteintech (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l hrp, by Beyotime (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l hrp, by Beyotime (1 mentions)
3 protocols using 60008 1 ig
1
Quantification of Adipogenic Proteins
2
Western Blot Characterization of OCC-1 in CRC
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For detection of endogenous OCC-1 polypeptide in CRC cells, western blot was performed according to the previous report in which the polypeptide was identified (31 (link)) using three commercially available primary antibodies (ab83945, ab83948 and ab177759, Abcam) raised against three different regions of human OCC-1 polypeptide.
For detection of other proteins in this study, western blot was performed according to standard methods. In brief, proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad) and incubated overnight at 4°C with corresponding antibodies: anti-FLAG M2-HRP (1:2000; A8592, Sigma), anti-GAPDH-HRP (1:30000; HRP-60004, proteintech), anti-ACTB (1:5000; 60008-I-Ig, proteintech), anti-HuR (1:1000; ab136542, Abcam) and anti-β-TrCP1 (1:1000; 1B1D2, 37–3400, Thermo Scientific). A HRP-conjugated sheep anti-mouse IgG secondary antibody (1:5000; SA00001-I, proteintech) was used for the detection of ACTB, endogenous HuR and β-TrCP1. The protein signals were detected using ECL chemiluminiscent substrate (FOREGENE).
For detection of other proteins in this study, western blot was performed according to standard methods. In brief, proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad) and incubated overnight at 4°C with corresponding antibodies: anti-FLAG M2-HRP (1:2000; A8592, Sigma), anti-GAPDH-HRP (1:30000; HRP-60004, proteintech), anti-ACTB (1:5000; 60008-I-Ig, proteintech), anti-HuR (1:1000; ab136542, Abcam) and anti-β-TrCP1 (1:1000; 1B1D2, 37–3400, Thermo Scientific). A HRP-conjugated sheep anti-mouse IgG secondary antibody (1:5000; SA00001-I, proteintech) was used for the detection of ACTB, endogenous HuR and β-TrCP1. The protein signals were detected using ECL chemiluminiscent substrate (FOREGENE).
3
Exosome Protein Extraction and Quantification
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RIPA lysis buffer (Thermo) was utilized to extract total protein from exosomes and HUVECs. BCA kit (Thermo Scientific, USA) was applied to determine total protein concentration. We used 10% SDS-PAGE to separate the samples, and then transferred samples to PVDF membranes. Subsequently, we utilized 5% milk to block the membranes at 24 °C temperature and treated with anti-CD63 primary antibodies (1:1000; Abcam, ab216130), CD9 (1:1000; Proteintech, 20597-I-AP), LFNG (1:5000; Abcam, ab151699), and β-actin (1:10,000; Proteintech, 60008-I-Ig) were incubated overnight at 4 °C. After washing 3 times with TBST buffer, the goat antirabbit IgG H&L (HRP) (1:1000; Beyotime, A0208) and goat antimouse IgG H&L (HRP) (1:1000; Beyotime, A0216) were incubated for 2 h at room temperature. Next, the high sensitive ECL luminescence kit was used for color development, the chemiluminescence imaging analysis system was exposed, and the pictures were collected. Finally, Image J (v5.2.1) was used for densitometric analysis.
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