After lysis with lysis buffer, 20 μg of protein per sample was loaded onto a lane of a sodium dodecyl sulphate-polyacrylamide gel for electrophoresis. The proteins were electrotransferred to polyvinylidene fluoride membranes. The transferred membrane was treated with blocking buffer and incubated with primary antibodies (E-cadherin [20874-1-AP; Proteintech, Chicago, IL, USA)], N-cadherin [22018-1-AP; Proteintech, Chicago, IL, USA], Cyclin D1 [60186-1-lg; Proteintech, Chicago, IL, USA], β actin [MAB1501R; Millipore, Burlington, VT, USA]) for 2 h at room temperature. Secondary antibodies (goat anti-rabbit [AP132P; Millipore, Burlington, VT, USA] and goat anti-mouse [AP124P; Millipore, Burlington, VT, USA]) were then added, and the cells were incubated for 90 min at room temperature. An enhanced chemiluminescence solution (205–14,621; Revvity, Burlington, VT, USA) was used to detect specific bands using a MINICHEMI (Thermo) system.
Minichemi
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MINICHEMI is a compact, benchtop chemistry analyzer designed for routine clinical chemistry testing. It provides automated analysis of a wide range of biochemical parameters from small sample volumes. The MINICHEMI is capable of performing photometric and ion-selective tests, offering a versatile solution for clinical laboratories and research applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Ap132p, by Merck Group (2 mentions)
Ap124p, by Merck Group (2 mentions)
β actin mab1501r, by Merck Group (1 mentions)
N cadherin 22018 1 ap, by Proteintech (1 mentions)
E cadherin 20874 1 ap, by Proteintech (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Pgc 1α, by Biorbyt (1 mentions)
Vascular endothelial growth factor (vegf), by Abcepta (1 mentions)
Cyclin d1, by Proteintech (1 mentions)
Spink1, by Abcam (1 mentions)
Stat3, by Santa Cruz Biotechnology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
A5441, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Anti n cadherin, by Proteintech (1 mentions)
4 protocols using minichemi
1
Western Blot Analysis of Cell Signaling Proteins
2
Protein Expression Analysis by Western Blot
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All samples were lysed in 200 μL lysis buffer. Then, 50 μg of protein for every sample was loaded for separation by SDS-PAGE at 50 V for 4 h. The protein was transferred from the gel to a PVDF membrane. After 1 h in blocking buffer, the membranes were incubated with primary antibodies [PGC1α (1:500; Biorbyt, Ely, UK; orb13647), β-actin (1:20,000; Sigma; A5441), N-cadherin (1:500; Proteintech, Rosemont, IL, USA; 22018-1-AP), E-cadherin (1:500; Proteintech, Rosemont, IL, USA; 20874-1-AP), cyclin D1 (1:500; Proteintech; 60186-1-lg), VEGF (1:500; ABGENT, San Diego, CA, USA; P15692)] for 2 h at room temperature and secondary antibodies [Goat anti-Rabbit (1:2000; Millipore, Billerica, MA, USA; AP132P) and Goat anti-Mouse (1:2000; Millipore; AP124P)] for 90 min. ECL solution (Western Lightning, Newton Abbot, UK; 205-14621) was used to detect specific bands with MINICHEMI (Thermo, Waltham, MA, USA).
3
Western Blot Analysis of Signaling Pathways
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All samples were prepared in 100 μl RIPA lysis buffer. 30 μg protein of every sample was loaded in the wells of SDS-PAGE with 80 V for 2 h then samples were transferred from the gel to the PVDF membrane with 400 mA for a further 2 h. After 1 h use of blocking buffer, the membranes were incubated with primary antibody SPINK1 (1:500; Abcam, USA, #ab58227), p-p38 (Cell Signaling, #9211), p38 (Cell Signaling, #9212), p-JNK (Cell Signaling, #4668), JNK (Cell Signaling, #9252), p-ERK (Cell Signaling, #9101), ERK (Cell Signaling, #9102), p-Akt (Cell Signaling, #9271S), AKT (Cell Signaling, #9272S), Stat3 (Santa Cruz, #sc-8019), p-PI3K (ABGENT, AP50389), PI3K (ABGENT, AP52796), β-actin (Sigma-Aldrich, MAB1501R), and secondary antibody Goat anti-Rabbit (1:2000; Millipore; AP132P) or Goat anti-Mouse (1:2000; Millipore; AP124P)] was applied. The signals were detected by ECL solution (Western Lightning; 205-14,621) with MINICHEMI (Thermo). Intensity of Western blot bands was digitally analyzed using ImageJ software.
4
Western Blot Protein Analysis Protocol
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The samples were lysed in 200 μL lysis buffer; 50 μg of samples were loaded into the wells of an SDS-PAGE system and electrophoresed at 50 V for 4 h. The proteins were transferred to PVDF membranes. After blocking membranes for 1 h, the membranes were incubated with the respective primary antibody. The antibodies used for western blotting included anti-NLRP12 (1:200; ABclonal, A6671), anti-N-cadherin (1:1000; Proteintech; 22018-1-AP), anti-VEGF (1:500; ABGENT; AP6290b), anti-E-cadherin (1:1000; Proteintech; 22874-1-AP), anti-βactin (1:20000; SIGMA; A5441), and anti-cyclin D1 (1:1000; Thermo; RM-9104). Membranes were incubated with the primary antibody for 2 h at room temperature, followed by incubation with the corresponding secondary antibody (goat anti-rabbit, 1:5000, Millipore, AP132P or goat anti-mouse, 1:5000, Millipore, AP124P) for 90 min. An enhanced chemiluminescence solution (Western Lightning; 205-14621) was used for detecting specific bands in a MINICHEMI (Thermo) system. Each western blot analysis was repeated thrice.
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