Total proteins extracted using a total protein extraction kit (Sangon Biotech) were quantified using a BCA protein assay kit (Sangon Biotech). Equal amount of protein was separated and transferred into PVDF membrane (Bio-Rad). The membranes containing the isolated proteins were blocked in 5% skim milk, exposed to the primary antibodies targeting LMO4 (ab229226; Abcam, Cambridge, MA, USA) and GAPDH (ab8245; Abcam) at 4°C overnight, and next reacted with the secondary antibody (ab205718; Abcam) for 2 h at room temperature. The indicated protein signals were presented using the enhanced chemiluminescence kit (Sangon Biotech) and quantified using the Image J software.
Enhanced chemiluminescence kit
Manufactured by Sangon
Sourced in China
The Enhanced chemiluminescence kit is a lab equipment product designed for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent substrate to produce a luminescent signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (2 mentions)
Ab8245, by Abcam (2 mentions)
Protein extraction kit, by Ipsen (2 mentions)
Hrp conjugated secondary antibody, by Abcam (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Bca protein assay kit, by Sangon (1 mentions)
Total protein extraction kit, by Sangon (1 mentions)
Ab229226, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab104836, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Anti ampk, by Abcam (1 mentions)
Anti apn, by Abcam (1 mentions)
Anti actb antibody, by Merck Group (1 mentions)
Anti foxo1a, by Abcam (1 mentions)
Anti p ampk, by Abcam (1 mentions)
Nonfat milk, by Solarbio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab6046, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
5 protocols using enhanced chemiluminescence kit
1
Quantitative Western Blot Analysis
2
Protein Expression Profiling via Western Blot
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Total proteins were isolated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into polyvinylidene difluoride (PVDF) membranes (Bio-Rad). After blocking by non-fat milk, the membranes were probed with the primary antibodies, including anti-Hexokinase 2 (HK2) (ab104836; Abcam), anti-Lactate dehydrogenase A (LDHA) (ab92903; Abcam), anti-TGFBR2 (ab204100; Abcam) and anti-GAPDH (ab8245; Abcam) at 4°C overnight, followed by the incubation of the secondary antibodies (ab205719; Abcam) at room temperature for 1 h. The protein signals were viewed using the enhanced chemiluminescence kit (Sangon Biotech).
3
Brain Protein Expression Analysis
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The expression levels of APN, APR1, APPL1, AMPK, pAMPK, FOXO1, and pFOXO1 in the brain and primary neurons were analyzed using Western blot analysis. Proteins from the brain tissues around the infarct area and primary neurons were collected using a protein extraction kit (Epizyme). Bicinchoninic acid assay was used to detect the concentration of proteins. An equal number of proteins in different groups were separated by Tris–glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Consequently, the membranes were blocked with 5% nonfat milk in PBST at room temperature for 1 h and incubated with primary antibodies (anti-APN, Abcam; anti-APR1, Proteintech; anti-APPL1, Proteintech; anti-AMPK, Abcam; anti-pAMPK, Abcam; anti-FOXO1A, Abcam; anti-pFOXO1A, Abcam; and anti-ACTB antibody, Sigma) at 4°C overnight. On the following day, the membranes were incubated with corresponding HRP-conjugated secondary antibody (Abcam) at room temperature for 1 h. The protein band was scanned on Amersham Imager 600 using an enhanced chemiluminescence kit (Sangon Biotech (Shanghai) Co.), and the relative amounts of proteins were analyzed using ImageJ software.
4
Tight Junction Protein Expression in Ischemic Brain
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The expression of tight junction (TJ) proteins in brain tissue were analyzed using western blot. At 24 h after surgery, brains were rapidly removed as mentioned above, and tissues around infarct area were collected to extract the whole proteins using the protein extraction kit (Epizyme). Bicinchoninic acid assay was used to determine the concentration of proteins. Equal amount of proteins (50 μg) in different groups were separated by Tris-glycine SDS page (6% and 10%) and transferred to nitrocellulose membrane. Consequently, the membranes were blocked with 5% non-fat milk in PBST at room temperature for 1 h and incubated with primary antibodies (ZO-1,absin, 1:1000; occludin, absin, 1:1000; claudin-5, absin, 1:1000; β-actin, sigma, 1:5000) at 4 °C overnight. The following day, the membranes were incubated with corresponding HRP-conjugated secondary antibody (abcam, 1:2000) at room temperature for 1.5 h. The protein band was scanned on Amersham Imager 600 using enhanced chemiluminescence kit (Sangon Biotech (Shanghai) Co.) and the relative amounts of proteins were analyzed using ImageJ software.
5
Quantification of Protein Expression
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Total protein was extracted from cells using RIPA buffer (Solarbio, China) mixed with 1% protease inhibitor PMSF (Solarbio, China). Once quantified by the BCA method, protein samples underwent SDS-PAGE separation and were transferred onto PVDF membranes (Millipore, USA). Following blocking with 5% non-fat milk (Solarbio, China), the membranes were left to incubate at 4 °C overnight with primary antibodies against FGF11 (1:1000; Abcam, ab89713, UK), HIF-1α (1:1000; Abcam, ab179483, UK), and β-tubulin (1:5000; Abcam, ab6046, UK). After washing with PBST, the membrane was incubated with an HRP-linked secondary antibody. An enhanced chemiluminescence kit was used to visualize the protein bands (Sangon, China). Image J (V2.0) was used to quantify the bands. Three independent experiments were performed for final analyses.
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