Total protein was extracted from ovaries using RIPA buffer (Cat. #P0013B, Beyotime, Shanghai, China) containing 1% protease inhibitor (MCE). Proteins were separated by 10% SDS-PAGE and transferred into polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies overnight at 4 ℃ after being blocked with Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat dry milk for 60 min. The following primary antibodies were used: Alix (ZENBIO, Chengdu, Sichuan, China), TSG101 (Proteintech, Wuhan, Hubei, China), CD9 (Proteintech), β-tubulin (Abbkine, Wuhan, Hubei, China), γH2AX (abcam, Boston, MA, USA), BAX (CST, Danvers, MA, USA), p53 (CST), Cleaved caspase-3 (CST), Bcl-2 (Abclonal, Wuhan, Hubei, China). Then, the membranes were washed three times with TBST for 10 min and incubated with secondary antibodies for 60 min. The signals were obtained through enhanced chemiluminescence (Thermo Fisher Scientific, Carlsbad, CA, USA) on the Tanon 5200 analysis system (Tanon, Shanghai, China).
β tubulin
Manufactured by Abbkine
Sourced in United States
β-tubulin is a structural protein that is a component of microtubules, which are cytoskeletal elements involved in various cellular processes such as cell division, intracellular transport, and cell motility. It plays a crucial role in the formation and function of the mitotic spindle during cell division.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Gapdh, by Abbkine (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by ABclonal (1 mentions)
γ h2ax, by Abcam (1 mentions)
Bcl 2, by ABclonal (1 mentions)
Tsg101, by Proteintech (1 mentions)
Cleaved caspase 3, by Merck Group (1 mentions)
α actin, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Enhanced chemoluminescence, by Beyotime (1 mentions)
Kisspeptin, by Abcam (1 mentions)
Peroxidase conjugated goat anti mice igg, by Jackson ImmunoResearch (1 mentions)
A01020, by Abbkine (1 mentions)
A01030, by Abbkine (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Cd206, by Proteintech (1 mentions)
β actin, by Abbkine (1 mentions)
6 protocols using β tubulin
1
Western Blot Analysis of Ovarian Proteins
2
Western Blot Analysis of Protein Targets
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Proteins were extracted using RIPA buffer and quantified using the BCA assay protocol. Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membranes. Samples were incubated with primary antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies. The blots were visualized using enhanced chemiluminescence and a Chemi-Doc Imaging System (Bio-Rad). Images were quantified and normalized using the ImageJ software (NIH). The primary antibodies used were UCP1 (Abcam), Caspase3 (Cell Signaling Technology), Cleaved caspase 3 (CST), α-actin (Sigma), and β-tubulin (Abbkine).
3
Immunoblotting Analysis of Mitochondrial Dynamics
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Cell lysate samples were prepared from cells in RIPA solution (Cat# FD009; Fude) supplemented with protease inhibitor (Cat# FD1001; Fude) and protein phosphatase inhibitor (Cat# FD1002; Fude). Denatured cell lysates were resolved by SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) filter membrane. After transfer, membranes were blocked in 5% (wt/vol) nonfat dry milk diluted in TBST. Membranes were incubated with primary antibodies against Mfn1 (1 : 1000, Cat# 14739; CST), Mfn2 (1 : 1000, Cat# 11925; CST), Drp1 (1 : 1000, Cat# 5391; CST), p-Drp1-S616 (1 : 500, Cat# 3455; CST), p-Drp1-S637 (1 : 500, Cat# 4867; CST), Opa1 (1 : 1000, Cat# ab157457; Abcam), PINK1 (1 : 1000, Cat# 6946; CST), NRF2 (1 : 1000, Cat# ab62352; Abcam), NLRP3 (1 : 1000, Cat# ab210491; Abcam), caspase 1 (1 : 1000, Cat# ab207802; Abcam), cleaved caspase 1 (P20) (1 : 1000, Cat# 4199; CST), PSMB5 (1 : 1000, Cat# abs115883; Absin), GAPDH (1 : 3000, Cat# FD0063; Fude), β-actin (1 : 1000, Cat# FD0060; Fude), and β-Tubulin (1 : 1000, Cat# A01030; Abbkine) overnight at 4°C and subsequently incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies which were detected by enhanced chemiluminescence (Cat# FD802; Fude). Immunoblots were analyzed using ImageJ 5.0 software (NIH; MD).
4
Western Blot Analysis of Hypothalamic Proteins
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Hypothalamus was homogenized in Radio Immunoprecipitation Assay Lysis buffer (RIPA) buffer and cleared by centrifugation, according to the standard techniques. Western blots of whole tissue lysates were probed with primary antibodies against Kisspeptin (ab226786, Abcam, Cambridge, United Kingdom), GnRH (PA5-97047, Thermo Fisher Scientific, Waltham, MA, United States), β-Tubulin (A01030, Abbkine, CA, United States), GAPDH (A01020; Abbkine, CA, United States). The secondary antibody used was either peroxidase-conjugated goat anti-rabbit IgG (111-035-003; Jackson, West Grove, PA, United States), or peroxidase-conjugated goat anti-mice IgG (115-035-003; Jackson, West Grove, PA, United States). Protein markers (20351ES76; Shanghai Yisheng, China) were added on both sides of each gel to verify bands. The polyvinylidene fluoride (PVDF) membranes were detected by enhanced chemoluminescence (Beyotime, China). The bands were analyzed using Image LabTM Software (Bio-Rab Laboratories, Hercules, CA, United States), were normalized to β-Tubulin or GAPDH, and were expressed as relative units (RU).
5
Protein Expression Analysis of Ovarian Tissue
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Ovaries were lysed in RAPI (P0013B, Beyotime Institute of Biotechnology, China) with 1% protease inhibitor (MCE, USA). Proteins were separated by electrophoresis by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred into polyvinylidene fluoride membranes (Bio‐Rad, USA). The membranes were blocked with 5% nonfat‐dry milk for 60 min and incubated at 4°C overnight with following primary antibodies: p‐RPS6 (9234, CST, USA), RPS6 (2217, CST, USA), p‐AKT (9271, CST, USA), AKT (9272, CST, USA), β‐tubulin (Abbkine, China), β‐actin (Abbkine), GAPDH (Abbkine), iNOS (60291, Proteintech) and CD206 (18704, Proteintech). The membranes were washed with TBST 10 min for three times and incubated with secondary antibodies for 60 min. The signals were enhanced through enhanced chemiluminescence (34094, Thermo Fisher Scientific, USA).
6
Western Blot Analysis of Brain Samples
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The brain samples
were homogenized in RIPA lysis buffer (Solarbio,
R0010) containing protease inhibitor tablets (4693159001, Roche) using
an automatic bead-based homogenizer (SAIERTE, China). The protein
was collected from the supernatant after centrifugation at 12,000
rpm for 15 min, the protein concentration was determined using the
BCA protein assay kit (Thermo Fisher Scientific), and proteins were
resolved on 10% SDS/PAGE gels. After transfer to 0.45 μm PVDF
membranes, the membranes were blocked for 1 h in blocking buffer and
then incubated overnight at 4 °C with the primary antibody (CD11b:
ab133357, Abcam; β-tubulin: A01030, Abbkine) in the same buffer.
The blots were then washed in TBS-T (TBS buffer containing 0.1% Tween-20),
incubated for 1 h with secondary antibodies (Goat Anti-Rabbit IgG
(H&L)-HRP: BE0101, EASYBIO; Goat Anti-Mouse IgG (H&L)-HRP:
BE0102, EASYBIO), and detected using a Tanon 5200 imaging system.
were homogenized in RIPA lysis buffer (Solarbio,
R0010) containing protease inhibitor tablets (4693159001, Roche) using
an automatic bead-based homogenizer (SAIERTE, China). The protein
was collected from the supernatant after centrifugation at 12,000
rpm for 15 min, the protein concentration was determined using the
BCA protein assay kit (Thermo Fisher Scientific), and proteins were
resolved on 10% SDS/PAGE gels. After transfer to 0.45 μm PVDF
membranes, the membranes were blocked for 1 h in blocking buffer and
then incubated overnight at 4 °C with the primary antibody (CD11b:
ab133357, Abcam; β-tubulin: A01030, Abbkine) in the same buffer.
The blots were then washed in TBS-T (TBS buffer containing 0.1% Tween-20),
incubated for 1 h with secondary antibodies (Goat Anti-Rabbit IgG
(H&L)-HRP: BE0101, EASYBIO; Goat Anti-Mouse IgG (H&L)-HRP:
BE0102, EASYBIO), and detected using a Tanon 5200 imaging system.
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