In order to determine protein concentration before the western blotting, a bicinchoninic acid (BCA) protein assay was performed. Next, equal protein samples were prepared with 4× laemmli and lysis buffer and boiled for 5 min. Samples were isolated on a 7.5% SDS‐PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was probed using primary antibody overnight at 4°C. Each blot was used to probe multiple antibodies, including anti‐rabbit NR2B (Abcam, ab254356, 1:1000), anti‐rabbit pNR2B‐Tyr1472 (ThermoFisher Scientific, 38–7000, 1:1000), anti‐rabbit p‐CREB (Abcam, ab32096, 1:5000), anti‐rabbit p‐AKT (Abcam, ab8805, 1:1500), anti‐rabbit p‐KAC (Cell Signaling Technology, 4781, 1:1000), anti‐mouse p‐CAMKII (Abcam, ab171095, 1:2000), anti‐rabbit p‐ERK (Abcam, ab229912, 1:1000), anti‐rabbit p‐MSK1 (Abcam, ab278550, 1:5000), and anti‐mouse actin (Sigma, A5316, 1:100,000) served as a loading control. The membrane was then incubated with horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse IgG secondary antibody for 1 h at room temperature. Lastly, the signal was detected using the Super Signal Chemi‐luminescent Substrate (Pierce).
Ab254356
Manufactured by Abcam
Ab254356 is a laboratory equipment product. It has a core function of performing a specific task related to scientific research or analysis. No further details about its intended use or features are provided in order to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab171095, by Abcam (2 mentions)
A5316, by Merck Group (2 mentions)
Ab32096, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab229912, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab124913, by Abcam (1 mentions)
Ab31232, by Abcam (1 mentions)
Ab52476, by Abcam (1 mentions)
Ab203663, by Abcam (1 mentions)
Ab133477, by Abcam (1 mentions)
Ab76321, by Abcam (1 mentions)
Ab109464, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
2 protocols using ab254356
1
Western Blot Protein Quantification
2
Western Blot Analysis of DRN Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Western blotting
was performed as previously described.68 (link) After completing behavior tests, mice were euthanized in the control,
MS, and O-1602 treatment groups (n = 3 per group,
7–8 weeks old). Subsequently, their brains were removed and
sliced, and the DRN was carefully excised under a microscope. Total
protein was extracted from DRN samples using the M-PER protein extraction
buffer. Protein concentration was determined using a BCA kit. Equal
amounts of protein samples were used for western blotting analysis.
The primary antibodies used in this experiment were as follows: GPR55
(Abcam, ab203663; 1:1000), GluN2A (Abcam, ab124913; 1:1000), GluN2B
(Abcam, ab254356; 1:1000), p-CaMKIIα (Abcam,
ab171095; 1:1000), CaMKIIα (Abcam, ab52476; 1:1000), p-GluA1R Ser831 (Abcam, ab109464; 1:1000), p-GluA1R Ser45 (Abcam, ab76321; 1:1000), GluA1 (Abcam, ab31232; 1:1000),
and TPH2 (Abcam, ab133477; 1:1000). β-Actin (Sigma-Aldrich,
A5316; 1:10,000) was used as a loading control. After three washes
with TBST for 10 min, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies. Then, the signal of the
target protein was detected and digitized using the ECL solution and
the ImageJ program. The band intensity of each blot was quantified
as a ratio relative to β-Actin. And we normalized all the data
to obtain the same value in the control group.
was performed as previously described.68 (link) After completing behavior tests, mice were euthanized in the control,
MS, and O-1602 treatment groups (n = 3 per group,
7–8 weeks old). Subsequently, their brains were removed and
sliced, and the DRN was carefully excised under a microscope. Total
protein was extracted from DRN samples using the M-PER protein extraction
buffer. Protein concentration was determined using a BCA kit. Equal
amounts of protein samples were used for western blotting analysis.
The primary antibodies used in this experiment were as follows: GPR55
(Abcam, ab203663; 1:1000), GluN2A (Abcam, ab124913; 1:1000), GluN2B
(Abcam, ab254356; 1:1000), p-CaMKIIα (Abcam,
ab171095; 1:1000), CaMKIIα (Abcam, ab52476; 1:1000), p-GluA1R Ser831 (Abcam, ab109464; 1:1000), p-GluA1R Ser45 (Abcam, ab76321; 1:1000), GluA1 (Abcam, ab31232; 1:1000),
and TPH2 (Abcam, ab133477; 1:1000). β-Actin (Sigma-Aldrich,
A5316; 1:10,000) was used as a loading control. After three washes
with TBST for 10 min, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies. Then, the signal of the
target protein was detected and digitized using the ECL solution and
the ImageJ program. The band intensity of each blot was quantified
as a ratio relative to β-Actin. And we normalized all the data
to obtain the same value in the control group.
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