The total protein was obtained using extraction kit (KGP250, KeyGen BioTECH, Nanjing, China), followed by the quantification using BCA protein concentration detection kit (KGA902, KeyGen BioTECH, Nanjing, China). By applying gel preparation kit (KGP113, KeyGen BioTECH, Nanjing, China), sodium dodecyl sulfate- (SDS-) PAGE was conducted followed by the membrane transfer. Thereafter, WB was performed, using anti-NIS (24324-1-AP, Proteintech Group, Inc., China), anti-Rap1GAP (ab32373, Abcam, UK), anti-TGF-β1 (ab215715, Abcam, UK), anti-Foxp3 (bs10211R, Bioss, China), anti-TβR1 (bs0638R, Bioss, China), anti-p-Smad3 (ab52903, Abcam, UK), anti-Smad3 (ab40854, Abcam, UK), anti-bcl-2 (ab182858, Abcam, UK), and anti-Bax (ab182733, Abcam, UK) with dilution rates of 1 : 1000, 1 : 10000, 1 : 1000, 1 : 1000, 1 : 1000, 1 : 2000, 1 : 1000, 1 : 2000, and 1 : 2000, respectively. After incubation with the secondary antibody, the membrane was colorized and imaged using G: BOX chemiXR5 (syngene, UK).
Kga902
Manufactured by Keygen Biotech
Sourced in China
The KGA902 is a high-performance laboratory centrifuge designed for a variety of applications. It features a compact and durable construction, capable of handling sample volumes up to 100 mL. The centrifuge offers a wide range of speed settings, reaching up to 6,000 RPM, and can maintain precise temperature control. The intuitive user interface allows for easy programming and monitoring of the centrifugation process.
Automatically generated - may contain errors
Lab products found in correlation
Kgp250, by Keygen Biotech (2 mentions)
Ab215715, by Abcam (1 mentions)
Ab52903, by Abcam (1 mentions)
Ab32373, by Abcam (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab40854, by Abcam (1 mentions)
Bs0638r, by Bioss Antibodies (1 mentions)
G box chemi xr5, by Syngene (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Ab68153, by Abcam (1 mentions)
Ab37185, by Abcam (1 mentions)
Ab137550, by Abcam (1 mentions)
Ab76315, by Abcam (1 mentions)
Ab31163, by Abcam (1 mentions)
Kgaa002, by Keygen Biotech (1 mentions)
Ab179463, by Abcam (1 mentions)
Ab182651, by Abcam (1 mentions)
Ab191606, by Abcam (1 mentions)
Kgaa37, by Keygen Biotech (1 mentions)
5 protocols using kga902
1
Quantitative Protein Analysis and Western Blotting
2
Western Blot Analysis of Key Apoptosis Regulators
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Total proteins were extracted after 48 h of incubation, and the concentrations were measured using the BCA method (KGA902, KeyGEN BioTECH, China). Then, the proteins were separated by SDS–PAGE and transferred onto NC membranes (Millipore, Bedford, MA, USA); the membranes were blocked with skim milk dissolved in TBST for 1 h at room temperature. The membranes were incubated with primary antibodies at 4°C overnight and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature after washing with TBST three times. After washing, the protein bands were detected with ECL substrate (KGP1201, Co Ltd, KeyGEN BioTECH, China). The bands were photographed by the G: BOX chemi XR5 system (Syngene Tech, Cambridge, UK). The primary antibodies used in this experiment and the relevant dilutions are described in Table 1 .
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Primary Antibodies Applied in This Work.
| Gene | Manufacturer | Cat. No. | Dilution | MW |
|---|---|---|---|---|
| Bcl-2 | Abcam plc, Cambridge, UK | ab287160 | 1:1000 | 20KD |
| TRAF1 | Proteintech Group Inc, Chicago, USA | 26845-1-AP | 1:1000 | 46KD |
| TP63 | Proteintech Group Inc, Chicago, USA | 60332-1-Ig | 1:500 | 68KD |
| BIRC3 | Proteintech Group Inc, Chicago, USA | 24304-1-AP | 1:2000 | 68KD |
| GAPDH | KeyGEN BioTECH, China | KGAA0002 | 1:1000 | 37KD |
3
Protein Expression Analysis of Lung Tissues
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Lung tissues and cells were homogenized and incubated in lysis buffer containing a protease inhibitor cocktail. The protein concentration was measured using a BCA kit (KGA902, KeyGEN BioTECH, Nanjing, China), and the proteins were then denatured at 100°C for 5 min. The proteins were loaded onto a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The blots were blocked with 5% nonfat milk at room temperature for 1 h and incubated with primary antibodies overnight at 4°C. The primary antibodies included rabbit monoclonal anti-Nrf2 (ab137550, Abcam, 1 : 1,000), anti-SLC7A11 (ab37185, Abcam, 1 : 1,000), anti-STAT3 (ab68153, Abcam, 1 : 2,000), anti-pSTAT3 (ab76315, Abcam, 1 : 2,000), and anti-β-actin (4970S, Cell Signaling Tech, 1 : 1,000). After washing three times with TBST for 15 min, the strips were incubated with anti-mouse or anti-rabbit horseradish peroxidase- (HRP-) conjugated secondary antibodies and detected using an ECL detector. The signals were scanned and quantified using ImageJ software.
4
Western Blot Analysis of Antioxidant Proteins
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Lung tissues were homogenized and incubated in lysis buffer containing a protease inhibitor cocktail. The protein concentrations were measured using a BCA kit (KGA902, KeyGEN BioTECH, Nanjing, China), and proteins were then denatured at 100 °C for 5 min. Proteins were loaded onto a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. Blots were blocked at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight. The details of primary antibodies used in this paper were shown as follows: rabbit anti-GPX4 (14432–1-ap, Proteintech, 1:500), rabbit anti-Nrf2 (ab31163, abcam, 1:500), mouse anti-HO1 (sc-136,960, santa, 1:200), and mouse anti-NQO1 (sc-32,793, santa, 1:200), rabbit anti-GAPDH (KGAA002, KeyGEN BioTECH, China, 1:10000). After washing, blots were incubated with fluorescence-labeled secondary antibodies.
5
Immunoblotting Analysis of PI3K/Akt Pathway
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Cellular proteins were extracted using a whole-protein extraction kit (KGP250, Keygen). Total protein was measured using a micro bicinchoninic acid protein determination kit (KGA902, Keygen). Thirty micrograms of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Then electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% milk in Tris-buffered saline plus Tween at room temperature for 2 h, then incubated at 4 °C overnight with primary antibody (protein kinase B [Akt], 1:1000 dilution, ab179463, Abcam; p-Akt, 1:5000 dilution, ab81283, Abcam; phosphatidylinositol 3-kinase [PI3K], 1:1000 dilution, ab191606, Abcam; p-PI3K, 1:1000 dilution, ab182651, Abcam) in 1% milk in Tris-buffered saline plus Tween. Finally, they were incubated for 2 h with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (KGAA37, Keygen). Protein bands were detected using enhanced chemiluminescent reagents (KGP116, Keygen).
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