Preparation of heparin media and analyses of EL overexpression were performed as described [22 (link)]. HDL-associated proteins (10 μg) and cell lysates (30 μg) were separated by 12% SDS-PAGE at 175 V for 90 min. Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Carl Roth, Karlsruhe, Germany) with blotting buffer (Tris, glycine, EDTA, sodium azide) at 150 mA for 90 min. Membranes were blocked at RT in 10% skim milk for 2 h followed by overnight incubation at 4 °C with antibodies specific for: human PON1 (Abcam, ab24261, Cambridge, UK), human apoA-I (Novus biological, NB100–65491, Littleton, CO, USA, or Academy biomedical, 11A-G2b, Houston, TX, USA), human LCAT (Novus biological, Littleton, CO, USA), human PAF-AH (Cayman chemical, 160603, Ann Arbor, MI, USA), α-tubulin (Cell signaling technology, 11H10, Leiden, Netherlands) and albumin (Abcam, ab 83465, Cambridge, UK). After washing and incubation with the appropriate secondary antibody (Dako, Vienna, Austria), protein signals were visualized by incubation with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Schwerte, Germany) using the ChemiDoc system (Bio-Rad Laboratories, Vienna, Austria).
Ab 83465
Manufactured by Abcam
Sourced in United Kingdom
Ab 83465 is a primary antibody used for the detection of a specific protein target. The product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Ab24261, by Novus Biologicals (2 mentions)
Human lcat, by Novus Biologicals (2 mentions)
Human paf ah, by Cayman Chemical (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc system, by Bio-Rad (2 mentions)
Ab53165, by Abcam (1 mentions)
Ecl luminol reagent, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
3 protocols using ab 83465
1
Western Blot Analysis of HDL-Associated Proteins
2
Vaccinia Virus Expression Analysis
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After 6 rounds of plaqued selection, 9 selected vaccinia VG9/(SST-14)2-HSA were propagated in BSC-40 cells. Then, the infected cells and supernatant were harvested and lysed through 3 freeze-thaw cycles. The lysates of vaccinia were separated by 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 3% BSA and incubated with primary antibodies against HSA (ab83465, Abcam, London, UK), somatostatin (ab53165, Abcam) and β-actin (Santa Cruz, CA, USA). Finally, corresponding HRP-conjugated anti-IgG secondary antibodies were incubated and the blots were developed by an ECL luminol reagent (Santa Cruz).
3
Western Blot Analysis of HDL-Associated Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Preparation of heparin media and analyses of EL overexpression were performed as described [22 (link)]. HDL-associated proteins (10 μg) and cell lysates (30 μg) were separated by 12% SDS-PAGE at 175 V for 90 min. Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Carl Roth, Karlsruhe, Germany) with blotting buffer (Tris, glycine, EDTA, sodium azide) at 150 mA for 90 min. Membranes were blocked at RT in 10% skim milk for 2 h followed by overnight incubation at 4 °C with antibodies specific for: human PON1 (Abcam, ab24261, Cambridge, UK), human apoA-I (Novus biological, NB100–65491, Littleton, CO, USA, or Academy biomedical, 11A-G2b, Houston, TX, USA), human LCAT (Novus biological, Littleton, CO, USA), human PAF-AH (Cayman chemical, 160603, Ann Arbor, MI, USA), α-tubulin (Cell signaling technology, 11H10, Leiden, Netherlands) and albumin (Abcam, ab 83465, Cambridge, UK). After washing and incubation with the appropriate secondary antibody (Dako, Vienna, Austria), protein signals were visualized by incubation with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Schwerte, Germany) using the ChemiDoc system (Bio-Rad Laboratories, Vienna, Austria).
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