The largest database of trusted experimental protocols

3 protocols using AMAb90618

Cells and tissues were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific), followed by sonication and centrifugation (10 min at 12,000 rpm at 4°C). Protein concentration was determined by the BCA method. Proteins were separated on Bolt™ 4–12% Bis-Tris Plus gels (Invitrogen, Thermo Fisher Scientific), blotted onto a nitrocellulose membrane (926–31092, LI-COR) and detected using the following primary antibodies: anti-ACOX1 (ab184032, Abcam), anti-ACAA1 (12319–2-AP, Proteintech), anti-EHHADH (GTX81126, Genetex), anti-HSD17B4 (ab128565, Abcam), anti-HMGCR (AMAb90618, Atlas Antibodies), anti-citrate synthase (GTX628143, Genetex), and anti-α-tubulin (32–2500, Thermo Fisher). Anti-MVK and anti-PMVK antibodies were a gift from Dr. Hans Waterham (AMC, Amsterdam) [36 (link), 37 (link)]. Proteins were visualized with goat anti-mouse and goat anti-rabbit secondary antibodies IRDye 800CW and IRDye 680RD (LI-COR, 926–32 210, 926–68 070, 926–32 211, 926–68 071). Band intensities were quantified using the Fiji distribution of ImageJ 1.x [38 (link)].
+ Open protocol
+ Expand
Livers were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific) using a TissueLyser II (Qiagen), followed by sonication and centrifugation (10 min at 12,000 rpm at 4°C). Protein concentration was determined by the BCA method. Proteins were separated on Bolt™ 4-12% Bis-Tris Plus gels or 7% Tris-Acetate gels (Invitrogen, Thermo Fisher Scientific), blotted onto a nitrocellulose membrane (926-31092, LI-COR) and detected as previously described (Ranea-Robles et al. 2021b (link)). The following primary antibodies were used: anti-ABCD3 (PA1-650, Invitrogen) anti-ACOX1 (ab184032, Abcam), anti-EHHADH (GTX81126, Genetex), anti-CROT (NBP1-85501, Novus Bio), anti-CPT2 (26555-1-AP, Proteintech), anti-MCAD (55210-1-AP, Proteintech), anti-CYP4A10 (PA3-033, Invitrogen), anti-HMGCR (AMAb90618, Atlas Antibodies), anti-phospho-ACC (Ser79, 3661, Cell Signaling), anti-ACC (3662, Cell Signaling), anti-MLYCD (SAB2702043, Sigma), and anti-α-tubulin (32-2500, Thermo Fisher). The anti-MVK antibody was a gift from Dr. Hans Waterham (AMC, Amsterdam) (Hogenboom et al. 2004a (link), b (link)).
+ Open protocol
+ Expand
3

Quantitative Western Blot Analysis of Lipid Metabolism Enzymes

Cells and tissues were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific), followed by sonication and centrifugation (10 min at 12,000 rpm at 4°C). Protein concentration was determined by the BCA method. Proteins were separated on Bolt TM 4-12% Bis-Tris Plus gels (Invitrogen, Thermo Fisher Scientific), blotted onto a nitrocellulose membrane (926-31092, LI-COR) and detected using the following primary antibodies: anti-ACOX1 (ab184032, Abcam), anti-ACAA1 (12319-2-AP, Proteintech), anti-EHHADH (GTX81126, Genetex), anti-HSD17B4 (ab128565, Abcam), anti-HMGCR (AMAb90618, Atlas Antibodies), anti-citrate synthase (GTX628143, Genetex), and anti-α-tubulin (32-2500, Thermo Fisher). Anti-MVK and anti-PMVK antibodies were a gift from Dr. Hans Waterham (AMC, Amsterdam) (Hogenboom et al. 2004b,a) . Proteins were visualized with goat anti-mouse and goat anti-rabbit secondary antibodies IRDye 800CW and IRDye 680RD (LI-COR, 926-32 210, 926-68 070, 926-32 211, 926-68 071) . Band intensities were quantified using the Fiji distribution of ImageJ 1.x (Schindelin et al. 2012) .
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!