Cell lysates were prepared as above followed by electrophoresis of the proteins on SDS-PAGE, with consequent transport onto PVDF membranes. Blots underwent blocking (120 min; ambient temperature) using 5% skimmed milk within TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween 20) before night-time incubation (4 °C) with anti-GOLGA3 (21193-1-AP, Proteintech, Waltham, MA, USA; 1: 5000) together with anti-p-CDK1(ab201008, Abcam, UK; 1: 5000). Following three TBS-T washing cycles, proteomic ontent samples were exposed (2 h, room temperature) to 2° antibodies (1:5000), evaluated visually through High-sig ECL® Western Blotting Substrate (Tanon, China).
Ab201008
Manufactured by Abcam
Sourced in United Kingdom
Ab201008 is a lab equipment product. It is a monoclonal antibody that can be used for research purposes. No further details about its core function or intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab32147, by Abcam (2 mentions)
Ecl kit, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab181861, by Abcam (1 mentions)
Ab209328, by Abcam (1 mentions)
Ab75908, by Abcam (1 mentions)
Ab32136, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab32034, by Abcam (1 mentions)
G 21234, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl detection kit, by Cytiva (1 mentions)
Ab32389, by Abcam (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Ab109520, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
5 protocols using ab201008
1
Immunoblotting Analysis of GOLGA3 and p-CDK1
2
Quantifying Spinal Cord Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A spinal cord segment, 0.5 cm in length, from where the injured site was centered
was used for protein quantification using western blotting. Proteins of the
nucleus and cytosol were extracted using the NE-PER Nuclear and Cytoplasmic
Extractions Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the
manufacturer’s instructions. The protein mixture extracted from each segment was
separated via electrophoresis and transferred to the polyvinylidene difluoride
membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were first
incubated with PFKFB3 (1:5,000, ab181861, Abcam, Cambridge, UK), Bax (1:1,000,
ab32503, Abcam), Bcl-2 (1:500, ab59348, Abcam), cleaved caspase-3 (CC-3, 1:500,
ab13847, Abcam), p-CDK1 (1:1,000, ab201008, Abcam), p27 (1:5,000, ab32034,
Abcam), H3 (1:5,000, ab1791, Abcam), myelin basic protein (MBP) (1:1,000,
ab209328, Abcam), APP (1:1,000, ab32136, Abcam), β-actin (1:5,000, ab8226,
Abcam), and p-p27 (1:1,000, ab75908, Abcam) antibodies at 4°C overnight,
respectively, and incubated with the secondary antibodies (1:10,000, ZB-2301 or
ZB-2305, Zhongshan Gold Bridge, Beijing, China) at 25°C for 1 h. The proteins
were detected using an ECL kit (Immobilon, Millipore, Billerica, MA, USA). The
gray values of each band were calibrated according to the internal reference and
compared to those of the sham group to acquire a relative value.
was used for protein quantification using western blotting. Proteins of the
nucleus and cytosol were extracted using the NE-PER Nuclear and Cytoplasmic
Extractions Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the
manufacturer’s instructions. The protein mixture extracted from each segment was
separated via electrophoresis and transferred to the polyvinylidene difluoride
membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were first
incubated with PFKFB3 (1:5,000, ab181861, Abcam, Cambridge, UK), Bax (1:1,000,
ab32503, Abcam), Bcl-2 (1:500, ab59348, Abcam), cleaved caspase-3 (CC-3, 1:500,
ab13847, Abcam), p-CDK1 (1:1,000, ab201008, Abcam), p27 (1:5,000, ab32034,
Abcam), H3 (1:5,000, ab1791, Abcam), myelin basic protein (MBP) (1:1,000,
ab209328, Abcam), APP (1:1,000, ab32136, Abcam), β-actin (1:5,000, ab8226,
Abcam), and p-p27 (1:1,000, ab75908, Abcam) antibodies at 4°C overnight,
respectively, and incubated with the secondary antibodies (1:10,000, ZB-2301 or
ZB-2305, Zhongshan Gold Bridge, Beijing, China) at 25°C for 1 h. The proteins
were detected using an ECL kit (Immobilon, Millipore, Billerica, MA, USA). The
gray values of each band were calibrated according to the internal reference and
compared to those of the sham group to acquire a relative value.
3
Protein Extraction and Western Blotting Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extraction of total proteins from HCCLM3 cells was carried out by the use of an ice-cold RIPA buffer lysis (Beyotime Biotechnology, Shanghai, China). The concentration of the extracted proteins was measured with BCA protein assay kit (Sigma-Aldrich, St. Louis, MO, USA). 20 µg of protein samples were separated with the application of SDS-PAGE, followed by a transferring performed on PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against DEPDC1B (Thermo Fisher Scientific, PA5-113,277, 1:500), cyclin-dependent kinase (CDK)1 (Abcam, ab201008, 1:1,000), CyclinD1 (Abcam, ab16663, 1:200), KIF23 (Thermo Fisher Scientific, A300-010A, 1:2,000), p53 (Abcam, ab32389, 1:10,000), p21 (Abcam, ab109520, 1:10,000) and GAPDH (Abcam, ab181602, 1:10,000) as the reference. Then the blots were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (Thermo Fisher Scientific, G-21234, 1:100,000). An enhanced chemiluminescence (ECL) detection kit (Cytiva, Shanghai, China) was adopted to develop these images of the protein bands. Finally, the quantification of bands was conducted with the help of Image J software (National Institutes of Health, Bethesda, MA, USA) [16 (link)].
4
Western Blot Analysis of Cell Cycle Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer was used to extract protein, and the protein concentration was quantified using the BCA protein concentration determination kit. The proteins (50 μg per sample) were resolved by electrophoresis under 100 V constant pressure and subsequently electro-transferred onto a PVDF transfer membrane. Then, the membrane was incubated in 5% bovine serum albumin (BSA) in TBST buffer at room temperature for 2 h, followed by incubation at 4 °C overnight with the primary antibodies as follows: anti-β-actin (1:5000; ab179467, Abcam), anti-CDC2 (1:1000; ab201008, Abcam), anti-CDK2 (1:1000; ab32147, Abcam), anti-Cyclin B1 (1:2000; ab181593, Abcam), anti-P21 (1:1000; ab188224, Abcam), anti-P27 (1:2000; ab193379, Abcam), anti-Wnt1 (1:500; 27935-1-AP, Proteintech), anti-β-catenin (1:500; ab68183, Abcam), anti-PRC1 (1:5000; ab51248, Abcam), and anti-NUF2 (1:1000; ab230313, Abcam). All antibodies were diluted with a TBST buffer containing 5% BSA. Next, the membranes were washed three times with TBST and incubated with secondary antibodies for 1 h. The membranes were then incubated with ECL chemiluminescence, and the bands were observed using a UVP chemiluminescence imaging system. We used β-actin protein as the internal reference protein to calculate the expression of the protein to be tested.
5
Immunohistochemical Analysis of Cell Cycle Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC was performed in paraffin-embedded sections using IHC kit (SV0002, BOSTER, China). Briefly, after being deparaffinized using xylene, and rehydrated using an ethanol gradient, the sections were incubated with 5% bovine serum albumin for 30 min at 37°C. Sections were probed with WDR5 (ab245479, Abcam, USA), CDK1 (ab201008, Abcam, USA), CDK2 (ab32147, Abcam, USA), CDK4 (ab68266, Abcam, USA), MMP2 (ab86607, Abcam, USA), and MMP9 (ab237782, Abcam, USA) overnight at 4°C, followed by incubation with the secondary antibody provided in the kits for another 30 min. The section was then stained with 3,3ʹ- diaminobenzidine, and was photographed by an Olympus inverted microscope using a ×20 objective.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!