The total protein extraction and western blot were performed, as previously described17 (link). The primary antibodies were ANXA5 (1:3000, Proteintech, China), p-ERK1/2 (Thr202/Tyr204) (1:1000, Cell Signaling Technology, USA), p-MEK (Ser217 /221) (1:1000, Cell Signaling Technology, USA), DOCK180 (1:400, Proteintech, China), RAC1 (1:800, Proteintech, China), C-MYC (1:800, Proteintech, China), α-Tubulin (1:5000, Proteintech, China), CRKI/II (1:1000, GeneTex, USA). Protein bands were visualized by ECL (Adavansta, USA) and imaged by Bio-Rad ChemiDocTM MP system (Bio-Rad, USA).
Anxa5
Manufactured by Proteintech
Sourced in China
ANXA5 is a recombinant protein that belongs to the annexin family. Annexins are a group of calcium-dependent phospholipid-binding proteins involved in various cellular processes. ANXA5 has the ability to bind to phosphatidylserine, a component of the cell membrane, and is often used as a marker for apoptosis detection.
Automatically generated - may contain errors
Lab products found in correlation
Crki 2, by GeneTex (2 mentions)
P erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
P mek ser217 221, by Cell Signaling Technology (1 mentions)
Chemidoctm mp system, by Bio-Rad (1 mentions)
C myc, by Proteintech (1 mentions)
α tubulin, by Proteintech (1 mentions)
Nitrocellulose filter membrane, by GE Healthcare (1 mentions)
Pparg, by Proteintech (1 mentions)
Casp3, by Proteintech (1 mentions)
Odyssey fluorescence imaging system, by LI COR (1 mentions)
Lysis buffer, by Keygen Biotech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Biotin streptavidin hrp detection system, by ZSGB-BIO (1 mentions)
3 3 diaminobenzidine dab kit, by ZSGB-BIO (1 mentions)
Bx3 cbh microscope, by Olympus (1 mentions)
3 protocols using anxa5
1
Western Blot Analysis of Cell Signaling Proteins
2
Protein Extraction and Western Blot Analysis
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Proteins from myocardial and renal tissues were extracted with lysis buffer (Keygen, China) supplemented with phenylmethylsulfonyl fluoride, phosphatase inhibitors, and protease inhibitors. Equal amounts of the protein samples were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose filter membrane (GE Healthcare Life Sciences, UK). After blocking in 5% bovine serum for 2 h, the membranes were incubated at 4°C overnight with primary antibodies against caspase 3 (CASP3; 1:1,000, Proteintech, USA), albumin (ALB; 1:1,000, Affinity, USA), epidermal growth factor receptor (EGFR; 1:1,000, Affinity, USA), annexin A5 (ANXA5; 1:1,000, Proteintech, USA), peroxisome proliferator activated receptor γ (PPARG; 1:1,000, Proteintech, USA), and GAPDH (1:1,000, Proteintech, USA). Then the membranes were washed 3 times with Tween 20/Tris-buffered saline and incubated with the secondary antibody (LI-COR Biosciences, USA) for 2 h at room temperature. After washing 3 times with Tris-buffered saline, the fluorescence signal of the secondary antibody could be detected by the Odyssey fluorescence imaging system (LI-COR Biosciences, USA).
3
Quantitative Immunohistochemical Analysis of ANXA5, RAC1, CRKI/II, CD34, and VEGF-3 in HCC and Mouse Xenograft Models
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IHC assay was done to determine the expression changes of ANXA5 (Proteintech, China), RAC1 (Proteintech, China) and CRKI/II (GeneTex,USA) in HCC tissue array, and of CD34 and VEGF-3 (SantaCruz, USA) in primary tissues from Hca-P-ANXA5-shRNA1 and Hca-P-ANXA5-shControl-transplanted mice. Tissue sections were treated with biotin-streptavidin HRP detection systems (ZSGB-BIO, China) according to the manufacturer’s protocol. The images were visualized with 3,3′-diamino-benzidine (DAB) kit (ZSGB-BIO, China) under an BX3-CBH microscope (Olympus, Japan).
IHC immunoreaction intensity was rated into four grades, 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) as Score I. Moreover, based on the detected positively staining cells, DAB staining quantity of each sample was graded as 0 (none), 1 (1–10% cells per field), 2 (10–50%), 3 (51–75%) and 4 (>76%) as Score II. The multiplication of Score I by Score II ranged 0 to 12 was used for IHC immunoreactivity degree. The scores of 0–2, 3–5, 6–8, and 9–12 were considered as negative (−), weak (+), moderate (++) and strong (+++). IHC assay was scored by two independent experienced pathologists, separately.
IHC immunoreaction intensity was rated into four grades, 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) as Score I. Moreover, based on the detected positively staining cells, DAB staining quantity of each sample was graded as 0 (none), 1 (1–10% cells per field), 2 (10–50%), 3 (51–75%) and 4 (>76%) as Score II. The multiplication of Score I by Score II ranged 0 to 12 was used for IHC immunoreactivity degree. The scores of 0–2, 3–5, 6–8, and 9–12 were considered as negative (−), weak (+), moderate (++) and strong (+++). IHC assay was scored by two independent experienced pathologists, separately.
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