Intestinal proteins were extracted from shrimp using a nuclear and cytoplasmic protein extraction kit (R0050, Solarbio, Beijing, China) following the manufacturer’s instructions. Intestinal protein was extracted from at least three shrimp in each experiment to reduce the individual differences.
R0050
Manufactured by Solarbio
Sourced in China
R0050 is a laboratory equipment designed for general research and laboratory applications. It serves as a versatile tool for various experimental procedures. The core function of R0050 is to provide a reliable and consistent platform for data collection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab13533, by Abcam (1 mentions)
Ab137037, by Abcam (1 mentions)
Affinipure goat anti mouse igg, by Proteintech (1 mentions)
Af0863, by Affinity Biosciences (1 mentions)
C3601, by Beyotime (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions)
C3606, by Beyotime (1 mentions)
Df7198, by Affinity Biosciences (1 mentions)
Bf0591, by Affinity Biosciences (1 mentions)
Af5135, by Affinity Biosciences (1 mentions)
Bc3710, by Solarbio (1 mentions)
Tm4sf1, by Abcam (1 mentions)
P jak2, by Abcam (1 mentions)
Flag antibody, by Merck Group (1 mentions)
Pd l1, by Abcam (1 mentions)
Lamin b1, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Tubulin, by Abcam (1 mentions)
P stat3, by Abcam (1 mentions)
P6730, by Solarbio (1 mentions)
9 protocols using r0050
1
Shrimp Intestinal Protein Extraction
2
Shrimp Intestine Protein Extraction
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The intestine tissue was dissected from shrimp and washed in PBS three times. Protein extraction was carried out by using a nuclear and cytoplasmic protein extraction kit (R0050, Solarbio, Beijing, China) following the manufacturer’s instructions. At least three shrimp were used for protein extraction to eliminate individual differences.
3
Protein Extraction and Western Blotting for Subcellular Analysis
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We used the same method as before to perform Western blotting.37 Before Western blotting, we used a total protein extraction kit (BC3710, Solarbio), cellular mitochondrial isolation kit (C3601, Beyotime) or tissue mitochondrial isolation kit (C3606, Beyotime), and nuclear protein extraction kit (R0050, Solarbio) to extract total protein, mitochondrial protein or de‐mitochondrial cytoplasmic protein, and nuclear protein, respectively. The primary antibodies used were anti‐PARP‐1 (1:1000, DF7198, Affinity); anti‐AIF (1:1000, BF0591, Affinity); anti‐COX IV (1:1000, AF5468, Affinity); anti‐Histone H3 (1:1000, AF0863, Affinity); anti‐β‐actin (1:10,000, 66009‐1‐Ig, Protentech); anti‐LC3BI/II (1:1000, A5402, Affinity); anti‐PINK1 (1:200, PA5‐85930, Invitrogen); anti‐Parkin (1:200, 702785, Invitrogen); anti‐SIRT3 (1:1000, Affinity, AF5135); anti‐SOD2/MnSOD (acetyl K68) (1:5000, ab137037, Abcam); and anti‐SOD2/MnSOD (1:5000, ab13533, Abcam). The second antibodies used were HRP‐conjugated AffiniPure Goat Anti‐Mouse IgG (1:10,000, Proteintech) and HRP‐conjugated AffiniPure Goat Anti‐Rabbit IgG (1:10,000, Proteintech). Finally, PVDF molds were visualized on a Tanon 2500R gel imaging system (Tanon) using a developer (Tanon), and the band intensity was quantified using ImageJ 1.39V software.
4
Cellular Fractionation and Immunoprecipitation Assay
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The cellular fractions were obtained by utilizing the nuclear protein extraction kit (R0050, Solarbio Life Science). Typically, cells were initially broken down using cytoplasmic protein extraction buffer, and then nuclear extraction buffer was used to isolate the nucleus fraction. The analysis employed antibodies against PD-L1, Tubulin, Lamin B1, NUP43, IPO5, GAPDH, TM4SF1, JAK2, p-JAK2, STAT3, and p-STAT3 from Abcam (UK). Additionally, Flag antibodies from Sigma (St. Louis, MO, USA) and secondary antibodies against mouse or rabbit from Cell Signaling Technology were used. In immunoprecipitation experiments, protein lysates were subjected to incubation with anti-PD-L1, anti-IPO5, anti-Flag, anti-Myc, or the normal IgG antibody at a temperature of 4 °C for an extended period of time overnight, with continuous rotation. On the second day, the mixture was also cultured with protein A/G beads or M2 anti-Flag resin at room temperature for a duration of 2–3 h. After undergoing three rounds of washing with lysis buffer, the beads were subjected to boiling and thereafter proceeded with a western blot assay.
5
Shrimp Intestine Protein Extraction
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The intestine tissue was dissected from shrimp and washed in PBS three times. Protein extraction was carried out by using a nuclear and cytoplasmic protein extraction kit (R0050, Solarbio , Beijing, China) following the manufacturer’s instructions. At least three shrimp were used for protein extraction to eliminate individual differences.
6
Isolation and Analysis of Nuclear Proteins
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Nuclear protein was extracted using a nucleus isolation kit according to manufacturer’s instructions (Solarbio, Beijing, China, R0050). Briefly, 30 mg liver was homogenized with a glass Dounce homogenizer in 500 μL lysis buffer, supplemented with protease inhibitor mixture (Solarbio, Beijing, China, P6730). Lysate was centrifugated at 700× g for 10 min at 4 °C. Protein concentration in supernatant solution was determined and boiled with protein loading buffer at 95 °C. The denatured protein sample containing cytosolic proteins was used to run western blot analysis. The pellet containing the nucleus was dissolved using lysis buffer and mixed with medium buffer and centrifuged at 700× g for 10 min at 4 °C. The supernatant was then discarded and the pellet resolved using lysis buffer. The protein concentration was determined and the nuclear sample denatured with protein loading buffer at 95 °C.
7
Protein Quantification and Immunoblotting
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Samples of brain tissue or PC12 cells were homogenized in RIPA lysis buffer supplemented with phenylmethane-sulfonyl fluoride and phosphatase inhibitors. Nuclear proteins were extracted from tissue homogenates using a nuclear extraction kit (R0050; Solarbio Biotechnology). For determining protein levels and levels of phosphorylation, equal quantities of proteins (60 μg in vivo and 30 μg in vitro) were separated using 10-12% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% nonfat milk for 2 h at room temperature, protein blots were probed with primary antibodies, such as those against AMPKα, p-AMPKα, NLRP3, Nrf-2, TXNIP, heme oxygenase-1 (HO-1), MAP-2, MBP, Iba-1, TNF-α, IL-18, IL-6, IL-1β, β-actin and Lamin B1 (listed in Table S1). On the second day, membranes were incubated with HRP-Goat Anti-Rabbit IgG (1:5000, SA00001-2, Proteintech, Wuhan, China) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Bio-Rad, Hercules, CA). Lastly, densitometry values were analyzed using ImageJ software.
8
Comprehensive Protein Analysis Protocol
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The protein concentration in the supernatant was measured using a BCA protein assay (Beyotime, Shanghai, China). Protein from total cell lysates was extracted using RIPA buffer with a protease inhibitor cocktail (NO. C0001, TargetMol, Boston, MA, D r a f t USA) and phosphatase inhibitor cocktails (NO. C0002-C0004, TargetMol, Boston, MA, USA) at a proportion of 1:100, and proteins from the nucleus were extracted using a nuclear protein extraction kit (R0050, Solarbio). Samples were resolved by 8-10%
SDS-PAGE and transferred to PVDF membranes (Solarbio, Beijing, China).
Antibodies against PDGFRβ (1:1000, ab32570, Abcam), GLUT1 (1:100000, ab115730, Abcam), HK2 (1:1000, ab209847, Abcam), PFK1 (1:1000, ab181064, Abcam), HIF1α
(1:1000, ab51608, Abcam), LDHA (1:1000, ab101562, Abcam), β-actin (1:100000, AC026, ABclonal), c-Myc (1:1000, #13987, CST), PDK1 (1:1000, ab207450, Abcam),
Anti-PDGFRβ phosphor Y571 (ab218534, 1:1000, Abcam), PKM2 (ab150377, 1:1000, Abcam), Anti-phospho-ERK1/ERK2 (T202/T185) (1:1000, ab201015, Abcam), ERK1/2 (1:2000, A5029, Bimake), AKT (1:1000.A5525, Bimake), p-AKT (1:1000, A5030, Bimake), mTOR(ab32028, 1:1000, Abcam), p-mTOR (ab109268, 1:1000, Abcam) and PCNA (1:2000, A5324, Bimake) were used.
SDS-PAGE and transferred to PVDF membranes (Solarbio, Beijing, China).
Antibodies against PDGFRβ (1:1000, ab32570, Abcam), GLUT1 (1:100000, ab115730, Abcam), HK2 (1:1000, ab209847, Abcam), PFK1 (1:1000, ab181064, Abcam), HIF1α
(1:1000, ab51608, Abcam), LDHA (1:1000, ab101562, Abcam), β-actin (1:100000, AC026, ABclonal), c-Myc (1:1000, #13987, CST), PDK1 (1:1000, ab207450, Abcam),
Anti-PDGFRβ phosphor Y571 (ab218534, 1:1000, Abcam), PKM2 (ab150377, 1:1000, Abcam), Anti-phospho-ERK1/ERK2 (T202/T185) (1:1000, ab201015, Abcam), ERK1/2 (1:2000, A5029, Bimake), AKT (1:1000.A5525, Bimake), p-AKT (1:1000, A5030, Bimake), mTOR(ab32028, 1:1000, Abcam), p-mTOR (ab109268, 1:1000, Abcam) and PCNA (1:2000, A5324, Bimake) were used.
9
NF-kB Signaling in Astrocyte Response
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The T10 segment of the spinal cord was rapidly collected at 3 dpi (n = 6), and the ventral section was dissected for western blot analysis. Astrocytes were harvested at 48 h after co-culture (n = 6), and proteins from cytoplasm and nuclear fraction were separately extracted using a nuclear protein extraction kit (R0050; Solarbio). Forty micrograms of proteins was separated and transferred onto polyvinylidene fluoride membranes, which were blocked in blocking buffer for 2 h and then incubated with primary antibodies against p65 (1:1000; #8242; Cell Signaling Technology, Danvers, MA, USA), phosphorylated-p65 (1:1000; #3033; Cell Signaling Technology), p-IKBα(1:1000; #2859; Cell Signaling Technology), IKBα (1:1000; #4812; Cell Signaling Technology), GFAP (1:500; SAB4300647; Sigma), C3 (1:100; ab11887; Abcam), β-actin (1:1000; 20536-1-AP; Proteintech), and Histone H3 (1:1000; #9715; Cell Signaling Technology) at 4°C overnight. After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody, for 2 h at 25℃. Bands were developed using enhanced chemiluminescence and band intensity was quantified using ImageJ.
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