Western blotting was used to detect the protein levels of Olr1, Hdlbp, Ldlr, Srebp-1c, Lp-pla2, Abcg1, Abca1, and Gapdh in RAW264.7 cells. Cells were routinely lysed to extract proteins. The extracted proteins were quantified using a BCA protein quantification kit (Thermo, USA). Conventional electrophoresis, membrane transfer, and antigen blocking operations were performed. Primary antibodies against Olr1 (dilution 1:1000; DF6522, Affinity, Melbourne, Australia), Hdlbp (1:1000; ab133594, Abcam, Cambridge, MA, USA), Ldlr (1:1000; DF7696, Affinity), Srebp-1c (1:1000; AF4728, Affinity), Lp-pla2 (1:1000; MA5-33112, Invitrogen, Carlsbad, CA, USA), Abcg1 (1:1000; ab52617, Abcam), Abca1 (1:1000; ab66217, Abcam), and Gapdh (1:5000; AB0037, Abways, Shanghai, China) were added to the incubation and incubated overnight in a box at 4 °C. The next day, the secondary antibody was added after the primary antibody was rinsed. Imaging agent (Tianneng, Shanghai, China) was added to the PVDF membrane to stain proteins. The experiments were repeated three times.
Ab66217
Manufactured by Abcam
Sourced in United States
Ab66217 is a rabbit monoclonal antibody designed to detect the GAPDH protein. It is suitable for use in Western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab52617, by Abcam (3 mentions)
Ab133594, by Abcam (1 mentions)
Bca protein quantification kit, by Thermo Fisher Scientific (1 mentions)
Df6522, by Affinity Biosciences (1 mentions)
Ab0037, by Abways (1 mentions)
Ab32376, by Abcam (1 mentions)
Ab32502, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ab92544, by Abcam (1 mentions)
Ab17052, by Abcam (1 mentions)
Ab82814, by Abcam (1 mentions)
Ab96899, by Abcam (1 mentions)
Ab126649, by Abcam (1 mentions)
Ab41927, by Abcam (1 mentions)
Ab98952, by Abcam (1 mentions)
Filipin 3, by Cayman Chemical (1 mentions)
Ab150120, by Abcam (1 mentions)
Imaging system, by Bio-Rad (1 mentions)
6 protocols using ab66217
1
Western Blot Analysis of Lipid Metabolism Proteins
2
Western Blot Analysis of ABCA1, ABCG1, and PKC
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Protein was extracted from cells or tissues using RIPA buffer with protease inhibitors (Sigma). Then, 5 × protein loading buffer was added to the sample at a protein: buffer ratio of 4:1, and the sample was heated in a metal bath at 60 °C for 10 min. Protein electrophoresis was performed using the TGX FastCast acrylamide kit (BIO) and then transferred to PVDF membranes (Millipore, Bedford, MA, America). After blocking in 5% skim milk for 2 h, the membrane was incubated with primary antibody (1:1000 or 1:500) in a refrigerator at 4 °C overnight. GAPDH was used as the loading control. Next, the membrane was washed in (0.1%) (TBS-T) at least three times for 10 min each and incubated with horseradish peroxidation-enhanced secondary antibody (1:10,000) for 2 h. ECL Prime (G.E.) was used to test the protein bands according to the manufacturer's instructions. The antibodies used in the experiment were as follows: GAPDH (ProMab,20,035); ABCA1 (Abcam,ab66217); ABCG1 (Abcam,ab52617); and p-PKCα (Abcam, ab32502;PKCα (Abcam, ab32376).
3
Cholesterol Metabolism Regulation
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27-Hydroxycholesterol was provided by American Santa Cruz, and 27-OHC (10 mg) was completely dissolved in a suitable amount of ethanol, then divided equally in centrifuge tubes, with nitrogen gas blowing dry, and finally preserved at −80°C. The 27-OHC working solution contains 0.16% ethanol (v/v). Filipin III was from Cayman Chemical (#70440, Ann Arbor, MI, United States). Primary antibodies for Western blot included mouse anti-ABCA1 (Abcam, ab66217), rabbit anti-ABCG1 (Abcam, ab52617), mouse anti-Caveolin-1 (Abcam, ab17052), rabbit anti-Apolipoprotein E (Abcam, ab52607), mouse anti-LDLR (Millipore, MABS26), rabbit anti-ACAT1 (Abcam, ab168342), rabbit anti-CYP46A1 (Sigma–Aldrich, SAB2100523), mouse anti-KDEL (Santa Cruz, sc-58774), rabbit anti-flotillin (Abcam, ab41927), rabbit anti-SR-B1 (Abcam, 52629), mouse anti- N Cadherin (Abcam, ab98952), mouse anti-Aβ (Abcam, ab126649), and rabbit anti-LRP1 (Abcam, ab92544). Goat anti-mouse (#14709) and rabbit (#7074) biotinylated secondary antibodies were from Cell Signaling Technology. Antibodies for immunofluorescence included anti-CYP46A1 (Abcam, ab82814), goat anti-mouse (Abcam, ab150120), and goat anti-rabbit (Abcam, ab96899).
4
Protein Expression Analysis Protocol
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Cell lysates were extracted using RIPA lysis buffer (G2033, Servicebio). Total cell protein was normalized using the BCA method (23, 227, Thermo Fisher) and separated using 8-12% SDS-PAGE gel electrophoresis before being transferred onto a nitrocellulose membrane. After blocking with 5% milk for 1 h, the membrane was incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. Antibodies used were as follows: anti-HOXB2 (1:1000, AY2582, Abways), anti-Beta Actin (1:5000, AB0035, Abways), anti-Beta tubulin (1:5000, 10086-1-AP, Proteintech), anti-γH2A.X (1:1000, GB111841, Servicebio), anti-Chk2 (1:10000, CY5633, Abways), anti-phospho Chk2 Thr68 (CY8878, Abways), anti-Chk1 (1:1000, 2360, Cell Signaling Technology), anti-phospho Chk1 Ser345 ((1:1000, 2348, Cell Signaling Technology), anti-ABCA1 (1:1000, ab66217, ABCAm), anti-ABCG1 (1:1000, 13578-1-AP, Proteintech), anti-phospho Erk1/2 Thr202/Tyr204 (1:2000, 4370, Cell Signaling Technology), and anti-Erk1/2 (1:1000, 4695, Cell Signaling Technology). Enhanced chemiluminescence was conducted using an HRP substrate (WBKLS0500, Merck) and visualized by a Bio-Rad imaging system.
5
Immunostaining of Brain Sections
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The following antibodies were used for immunostaining of 50–100 µm thick floating brain sections: goat anti-ChAT, 1:1000 (AB143; Millipore, Billerica, MA); rabbit anti-GFP, 1:1000 (A11122; Invitrogen); mouse anti-β-amyloid (6E10), 1:500 (NE1003; Millipore); rabbit anti-GFAP, 1:1000 (AB5804; Millipore); rabbit anti-Iba1, 1:1000 (019-19741; Wako, Richmond, VA); mouse anti-GFAP 1:1000 (MAB360; Millipore); mouse anti-ABCA1 monoclonal antibody HJ1 (ab66217; Abcam, Cambridge, MA); mouse anti-β-Actin antibody AC-15 (A5441; Sigma); and rabbit anti-TH, 1:1000 (AB152; Millipore). Secondary antibodies were from Invitrogen. GS-lectin staining used Alexa488-IB4, 1:1000 (I21411; Invitrogen, Grand Island, NY). Brain sections were incubated in primary antibodies diluted in PBS, 0.5% Triton X-100, 0.1 mM CaCl2 (PBSTC) + 10% normal goat or donkey serum, washed in PBSTC for 6 hr, and incubated at 4°C overnight in secondary antibodies diluted in PBSTC +10% normal goat or donkey serum. After washing in PBSTC for 4–6 hr, brain sections were mounted in Fluoromount G (17984-25; EM Sciences, Hatfield, PA). Images were captured on a Zeiss LSM700 confocal microscope and processed with Zen software, ImageJ/Fiji, and Adobe Photoshop.
6
Western Blot Analysis of NF-κB Pathway Proteins
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Total protein was extracted from cultured cells and liver tissues by using ice cold RIPA lysis buffer. The protein concentration was determined by BCA protein assay kit (Solarbio, Beijing, China). 20 μg of protein was ran on a 10% SDS-PAGE gel and transferred to a PVDF (Millipore, USA) membrane. GAPDH was used as a control and non-specific bindings were blocked by 5% BSA for 40 min at room temperature. Incubates the specific primary antibodies against p-IKKβ (Abcam, ab59195, 1:1000), IKKβ (Abcam, ab124957, 1:1000), p-p65 (Abcam, ab86299, 1:1000 dilution), p65 (Abcam, ab16502, 1:1000 dilution), TNF-α (Abcam, ab183218, 1:1000 dilution), IL-6 (Abcam, ab259341, 1:1000 dilution), ABCA1 (Abcam, ab66217, 1:1000), CPT1 (Abcam, ab234111, 1:1000) at 4 ℃ overnight. After extensively washing with 100 µM PBST, peroxidase-conjugated secondary antibody (Abcam, ab205718, 1:5000) was applied to the membranes and incubated for 2 h at room temperature. Protein bands were detected by using ECL substrates (Thermal Fisher, Rockford, USA) and visualized under a ChemiDoc XRS + imager (Bio-Rad, Hercules, USA).
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