Total protein was isolated from cerebral cortex or microglia with homogenization in lysis buffer and centrifuged at 12,000 rpm about 15 min. The BCA kit (Cell Signaling Technology, Boston, MA) was used to determine the protein concentrations. The western blotting was conducted according to the previous description (Zhu et al., 2019 ). Then, the proteins were incubated overnight at 4℃ with specific primary antibodies, which including Iba‐1 (1:1,000; ab178846; Abcam), JNK (1:1,000; #9252; Cell Signaling Technology, MA), phosphorylated‐JNK (p‐JNK; 1:1,000; #9251; Cell Signaling Technology), P65 (1:1,000; #3039; Cell Signaling Technology), I‐kBa (1:1,000; #9242; Cell Signaling Technology), and subsequently detected using the secondary antibody (1:2,000; Cell Signaling Technology).
Bca kit
Manufactured by Cell Signaling Technology
Sourced in United States
The BCA kit is a colorimetric assay used to determine the total protein concentration in a sample. It is based on the reduction of copper ions by proteins in an alkaline environment, resulting in a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Iba 1, by Abcam (1 mentions)
Phosphorylated jnk p jnk, by Cell Signaling Technology (1 mentions)
Ab46172, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Ab2302, by Abcam (1 mentions)
Ab108319, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Cell Signaling Technology (1 mentions)
3 protocols using bca kit
1
Western Blot Analysis of Cerebral Cortex Proteins
2
Western Blot Analysis of Neuronal Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The neurons were lysed using RIPA buffer (#9806, Cell Signaling Technology, Danvers, MA, USA). Protein concentration was determined with a Bicinchoninic protein assay (BCA) kit (7780S, Cell Signaling Technology, Danvers, MA, USA). All the protein samples were then electrophoresed via 10% SDS-PAGE (P0012A Beyotime, China). Later, the samples were transferred to polyvinylidene fluoride membranes (PVDF; Millipore, Bedford, MA) which were blocked by 5% skimmed milk at room temperature for 60 min. The membranes were then incubated with the following primary antibodies overnight at 4ºC: anti-GAPDH antibody (rabbit, #5174, 1 : 1000, Cell Signaling Technology, USA), anti-NGF antibody (rabbit, ab221609, 1 : 1000, Abcam, UK), anti-C-caspase-3 antibody (rabbit, ab2302, 1 µg/ml, Abcam, UK), anti-CNTF antibody (rabbit, ab46172, 1 : 3000, Abcam, UK) and anti-BDNF antibody (rabbit, ab108319, 1 : 1000, Abcam, UK). The membranes were then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-rabbit IgG H&L (HRP) (ab205718, 1 : 2000, Abcam, UK) for 1 h at room temperature and washed with tris-buffer saline tween (TBST) three times. GAPDH was employed as the normalization reference. An enhanced chemiluminescence (ECL) plus kit (K22030, Abbkine Scientific, China) was employed to measure the labelled proteins [36] .
3
Western Blot Analysis of SMAD5 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MC3T3-E1 cells were lysed by cell lysis buffer containing the protease inhibitor PMSF (Cell Signaling Technology, MA, USA) and scratched on ice. The lysed buffer was centrifuged at 12000 r/min for 20 minutes, and the protein concentration in the supernatant was determined by BCA kit (Cell Signaling Technology, MA, USA). The protein samples were further prepared by mixing the same volume of loading buffer with 20 μg of total protein that had been denatured under 100°C boiling water. The proteins were then separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose blotting membrane under 200 mA for 60–90 minutes. The membrane was then incubated with primary rabbit anti-mouse SMAD5, p-SMAD5, or GAPDH antibody (1 : 1000; Abcam, Hongkang, China) under 4°C overnight after blocking with skim milk for 2 hours. Second goat anti-rabbit antibody conjugated with horseradish peroxidase (HRP) (1 : 5000; Abcam, Hongkang, China) was applied, and the membranes were incubated for 2 hours under room temperature. The protein banding was then determined using the enhanced chemiluminescence method (Cell Signaling Technology, MA, USA). The intensities of Western blot bands were determined, and protein expression was expressed as a ratio relative to GAPDH expression.
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!