Whole cell protein extracts prepared using the RIPA buffer (Beyotime Institute of Biotechnology) and quantified using the BCA Protein assay kit (Beyotime Institute of Biotechnology). Protein (20 µg per lane) was separated by 12% SDS-PAGE and then transferred to a nitrocellulose membrane (Pharmacia; GE Healthcare). After blocking with 5% skimmed milk at room temperature for 1 h, the membrane was incubated with diluted primary antibody at 4°C overnight. The primary antibodies used were anti-Pmel (1:300), anti-syntenin (1:500), anti-Tyr (1:200), anti-MITF (1:500), anti-β-actin (1:1,000), anti-p38 (1:500) and p-p38 (1:500). Then the membrane was incubated with HRP-labeled secondary antibodies at room temperature for 2 h. Protein bands were visualized using ECL reagents (Roche Diagnostics GmbH). Protein expression levels were semi-quantified using Gel-Pro Analyzer software (version 4.0; Media Cybernetics, Inc.) with β-actin as the loading control.
Pharmacia
Manufactured by GE Healthcare
Sourced in United States, Germany
Pharmacia is a line of laboratory equipment and instrumentation produced by GE Healthcare. It is designed for use in various research and diagnostic applications, including chromatography, electrophoresis, and protein purification. Pharmacia equipment is known for its precision, reliability, and versatility in supporting a wide range of laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Ecl reagent, by Roche (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
Streptomycin, by GE Healthcare (1 mentions)
Deae 52 cellulose, by Merck Group (1 mentions)
Trizol reagent, by Bio-Rad (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Sephadex g 100, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
Phastgel, by GE Healthcare (1 mentions)
4 protocols using pharmacia
1
Protein Expression Analysis in Melanocytes
2
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected and lysed in RIPA buffer containing protease inhibitors. 20 µg of protein were separated via SDS-PAGE on a 12% polyacrylamide gel in running buffer. After electrophoresis, the proteins were blotted onto nitrocellulose membranes (Amersham, Bensheim, Germany), after which the membranes were incubated in PBST containing 5% fat-free milk (Nestle, Beijing, China) for 1 h at room temperature, followed by incubation with different primary antibodies overnight at 4°C. The primary antibodies were detected using a horse-radish peroxidase-linked secondary antibody in conjunction with an enhanced chemiluminescence (ECL) reagent (Pharmacia; GE Healthcare, Chicago, IL, USA).
3
Astragalus membranaceus Root Extraction and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Astragalus membranaceus roots were purchased from Guangyi Chinese Herbal Cultivation Co., Ltd (Fengzhen, China). DEAE-cellulose 52 and Sephadex G-100 were purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany) and Pharmacia; GE Healthcare Life Sciences (Uppsala, Sweden), respectively. 3-(4 (link),5 )-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich; Merck Millipore. DMEM, streptomycin and penicillin were obtained from Hyclone; GE Healthcare Life Sciences (Logan, UT, USA). ELISA kits for TGF-β1, bFGF, EGF and cyclin D1 were purchased from Shanghai Honsun Biological Co., Ltd. (Shanghai China). TRIzol reagent and SYBR Green I detection reagents were purchased from Bio-Rad Laboratories, Inc., (Hercules, CA, USA). Bovine serum albumin (BSA) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The secondary antibody was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). All other reagents were of analytical grade and purchased from local chemical suppliers in China.
4
Desialylation and Leg5Ac7Ac Modification of α1-Antitrypsin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The glycoprotein (6 mg) was desialylated in MES buffer pH 6.0 (1 mL of 0.5 M) with 25 mU of M. viridifaciens sialidase, at 37 o C for 4 h. Removal of the sialic acid residues was confirmed by iso-electric focussing (IEF), carried out with a Pharmacia (GE Healthcare) Phastgel apparatus, using precast Phastgel pH 3-9, and manual staining with Coomassie Blue. The product was diluted to 50 mL with 20 mM Tris buffer pH 7.5 and purified by ion-exchange chromatography on a 5ml HiTrap Q HP column, run in the same Tris buffer and eluted with a 0-0.3M NaCl gradient.
The asialo-α1-antitrypsin was modified with Leg5Ac7Ac by incubation of 0.5 mL of the protein (3.03 mg/mL) with 100 µl of 0.5 M MES buffer, pH 6.5, 100 µL of 100 mM MgCl2, 300 µL of 10 mM CMP-Leg5Ac7Ac and 100 µL of ST6Gal1, for 20 h at 30 o C; a second 300 µL aliquot of the CMP-Leg5Ac7Ac was added after 4 h. The reactions were checked by IEF. The reaction mixture was diluted with 100 mL of 20 mM Tris buffer pH 7.5, and the product was purified by ion exchange as described above. The removal of sialic acid residues and the addition of Leg5Ac7Ac were quantified by mass spectrometry.
The asialo-α1-antitrypsin was modified with Leg5Ac7Ac by incubation of 0.5 mL of the protein (3.03 mg/mL) with 100 µl of 0.5 M MES buffer, pH 6.5, 100 µL of 100 mM MgCl2, 300 µL of 10 mM CMP-Leg5Ac7Ac and 100 µL of ST6Gal1, for 20 h at 30 o C; a second 300 µL aliquot of the CMP-Leg5Ac7Ac was added after 4 h. The reactions were checked by IEF. The reaction mixture was diluted with 100 mL of 20 mM Tris buffer pH 7.5, and the product was purified by ion exchange as described above. The removal of sialic acid residues and the addition of Leg5Ac7Ac were quantified by mass spectrometry.
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!