Cells were lysed in RIPA-buffer [1% Nonident P-40, 50 mmol/L Tris-HCl (pH 7.4), and 150 mmol/L NaCl] supplemented with protease inhibitors (Sigma). Protein lysates were subjected to 6% SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad), and incubated with anti-Enpp1 (PA5-17097, Invitrogen), anti-Epas1 (sc-13596, Santa Cruz, RRID:AB_627525), anti-Srpx2 (Ab01584, Abcam), anti-Apcdd1 (sc-84694, Santa Cruz, RRID:AB_2057638), anti-Hp (ab256454, Abcam), and anti-tubulin antibody (T4026, Sigma-Aldrich). Bands were developed with enhanced chemiluminescence system (Amersham Bioscience).
Ab256454
Manufactured by Abcam
Sourced in United States
Ab256454 is a laboratory equipment product. It is designed to perform a specific core function in the laboratory setting. The detailed description of the product's features and intended use is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Sc 13596, by Santa Cruz Biotechnology (2 mentions)
Pa5 17097, by Thermo Fisher Scientific (2 mentions)
Ab01584, by Abcam (2 mentions)
Sc 84694, by Santa Cruz Biotechnology (2 mentions)
T4026, by Merck Group (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Enhanced chemiluminescence system, by Cytiva (1 mentions)
β actin, by Applygen (1 mentions)
Ab58703, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ab185544, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Piercetm bicinchoninic acid bca assay, by Thermo Fisher Scientific (1 mentions)
A5258, by ABclonal (1 mentions)
As014, by ABclonal (1 mentions)
Cb1001, by Merck Group (1 mentions)
Ab15323, by Abcam (1 mentions)
As003, by ABclonal (1 mentions)
5 protocols using ab256454
1
Protein Expression Analysis Protocol
2
Quantification of Hippocampal Protein Levels
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Western blotting was performed with primary antibodies against 14-3-3β/α (#sc25276; 1:500; Santa Cruz, CA, USA), haptoglobin (#ab256454; 1:1000; Abcam, Cambridge, UK), S100A6 (#ab181975; 1:10,000; Abcam), ClpP (#sc271284; 1:1000; Santa Cruz), A2M (#ab58703; 1:1000; Abcam), BVR-A (#sc393385; 1:1000; Santa Cruz), and β-actin (#C1313-100 1:1000; Applygen, Beijing, China) in tissues from the hippocampus, prefrontal cortex, and temporal lobe. A horseradish peroxidase conjugated labeled secondary antibody was used. Immunoreactivity was visualized by scanning the membranes with an Odyssey infrared imaging system (LI-COR). All procedures were performed by experienced experimenters blinded to the treatment condition.
3
Protein Isolation and Western Blot Analysis
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The cells were treated under the indicated conditions and then efficient lysed with RIPA buffer at pH 8.0 (150 mM NaCl, 50 mM Tris, 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% SDS). All the samples were quantified using PierceTM bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Inc.) to ensure equal loading of proteins. The cellular protein samples were separated by SDS‐PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore). After blocked by 5% BSA, the membrane was probed with the primary antibody and then incubated with HRP‐conjugated secondary antibody in TBST. The primary antibodies include anti‐TXN2 (1:1000 dilution, Abcam, ab185544) and anti‐HP (1:1000 dilution, Abcam, ab256454).
4
Western Blot Analysis of Retinal Proteins
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The protein sample from the retina was denatured by boiling in SDS sample buffer, and an aliquot of 10ug of total protein was subjected to 10% SDS-PAGE gels, followed by transferring to a PVDF membrane. The membrane was first blocked with 5% non-fat milk in TBST buffer for one hour and then incubated overnight at 4 °C with primary antibodies. After washing in TBST for three times, the membrane was incubated in a blocking solution with secondary antibodies at RT for 1 h. The membranes were washed 5 times and incubated with SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol and were ready for detection. The antibodies and concentration used are listed below: rabbit anti-iNOS (ab15323, Abcam, 1:250), rabbit anti-TLR4 (A5258, Abclonal, 1:1000), rabbit anti-HP (ab256454, Abcam, 1:1000), rabbit anti-HPX (A5603, Abclonal, 1:1000), rabbit anti-FGG (A5642, Abclonal, 1:1000), mouse anti-GAPDH (CB1001, Millipore, 1:1000), HRP goat anti-rabbit IgG (AS014, Abclonal, 1:5000) and HRP goat anti-mouse IgG (AS003, Abclonal, 1:5000).
5
Western Blot Analysis of Protein Targets
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