Cells or tissue samples were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Proteintech, USA) with phenylmethylsulfonyl fluoride (PMSF) (1 mM, final concentration). Extracted proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were then incubated overnight at 4 °C in appropriate dilutions of anti-TTP rabbit polyclonal antibody (ab33058, Abcam) or anti-IL-33 mouse monoclonal antibody (ab54385, Abcam), followed by, respectively, goat anti-rabbit IgG or goat anti-mouse IgG (Proteintech, USA), as secondary antibodies. ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore, USA) was used for detection, and anti-β-actin antibody (Proteintech, USA) was used as an internal control.
Ripa lysis buffer
Manufactured by Proteintech
Sourced in United States
RIPA lysis buffer is a protein extraction and cell lysis reagent used for the preparation of protein samples from cells or tissues for various downstream applications, such as Western blotting, immunoprecipitation, and protein quantification.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Proteintech (1 mentions)
Anti β actin antibody, by Proteintech (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ab33058, by Abcam (1 mentions)
Ab54385, by Abcam (1 mentions)
Goat anti mouse igg, by Proteintech (1 mentions)
Protein a g plus agarose, by Proteintech (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Proclin 300, by Merck Group (1 mentions)
Polyvinylpyrrolidone k30, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Rabbit anti gapdh antibody, by Proteintech (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions)
Alpha tubulin monoclonal antibody, by Proteintech (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Hrp conjugated affinipure goat anti mouse igg, by Proteintech (1 mentions)
Gapdh 60004 1 lg, by Proteintech (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
E2f1 primary antibody, by Abcam (1 mentions)
6 protocols using ripa lysis buffer
1
Protein Expression Analysis by Western Blot
2
TGEV Interaction with UBXN1 in IPEC-J2
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The lysate of IPEC-J2 cells infected with TGEV Miller for 36 h was prepared with RIPA lysis buffer (Proteintech) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMSF) (1 mM). After centrifugation at 10 000 × g for 20 min and measurement of the protein concentration using the BCA method, the lysate supernatant was pretreated with protein A/G PLUS-Agarose (Proteintech) for 60 min at 4 °C to purify the protein. The lysate supernatant (700 μg) was incubated with 3 μg of a rabbit pAb against UBXN1 overnight at 4 °C. Next, 10 μL of protein A/G PLUS-Agarose was added to this mixture and incubated with shaking at 4 °C for 4 h. After washing four times with lysis buffer, the eluted proteins were analyzed by SDS-PAGE and Western blotting using pAbs recognizing the S1 protein of TGEV and rabbit pAbs recognizing UBXN1. The lysate of IPEC-J2 cells uninfected with TGEV was used as the control.
3
Cyfra 21-1 Immunoassay Development
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Anti-Cyfra 21-1 detecting antibodies, anti-Cyfra 21-1 capturing antibodies, and Cyfra 21-1 recombinant antigen were purchased from Shanghai LingChao Biological Co., Ltd. (Shanghai, China) and goat-anti-mouse IgG was purchased from ShengGong Biotech (Shanghai, China). Glass cellulose membranes (conjugate pad), polyester film (sample pad), absorption pads, and polyvinyl chloride (PVC) baseplates were also purchased from Shanghai JinBiao Biotechnology Co., Ltd. Nitrocellulose filter membranes were purchased from Life Sciences (California, United States). Chemical reagents, including gold chloride (HAuCl4·3H2O), sodium citrate (C6H5Na3O7·2H2O), sodium azide, ProClin 300, Tween 20, and polyvinylpyrrolidone K30 were purchased from Sigma (St. Louis, MO, United States). RIPA lysis buffer was obtained from Proteintech (Wuhan, China). A three-dimensional film cutting and gold spraying instrument, automatic cutting machine, shell pressing machine, and chromatography reader were purchased from Shanghai JinBiao Biotechnology Co., Ltd. (Shanghai, China).
4
Protein Expression Analysis in Neurological Disorders
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Human brain tissues from patients with TLE or traumatic brain injury, as well as rat cortices and hippocampi, were homogenized in RIPA lysis buffer (Proteintech, China). The supernatants were removed after centrifugation at 4°C (12,000 g, 15 min). Prepared proteins (40 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE; 5% spacer gel, 80 V, 30 min; 10% separating gel, 120 V, 90 min) before being transferred to a polyvinylidene fluoride (PVDF) membrane (220 mA, between 60 and 120 min according to the molecular weight of the protein). Next, the PVDF membrane was incubated at 37°C for 1 h in 10% skim milk in Tween-20 for blocking. The PVDF membrane was then incubated with polyclonal mouse anti-SAD-B-1 (diluted 1:100, Cat. No. ab206298; Abcam) diluted in 10% skim milk diluted in freshly prepared 5% BSA at 4°C overnight. The membrane was washed with Tween-20-Tris-buffered saline (TBS) for 45 min, with the solution changed every 10 min, and then incubated with goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody (1:1000, Proteintech, China) for 1 h at 37°C. A rabbit anti-GAPDH antibody (1:1000, Proteintech) was used as the loading control. Densitometry quantitation was determined using Quantity One 1-D Analysis Software (Bio-Rad Laboratories, USA) as optical density values, and the SAD-B levels were normalized to those of GAPDH.
5
Western Blot Analysis of Phospho-EIF2S1
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Cellular proteins were extracted using RIPA lysis buffer (Proteintech,Rosemont, IL, USA). Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore, USA). After blocking with 5% skimmed milk in TBST for 1 h, membranes were incubated with primary antibodies, including phospho-EIF2S1 (Ser51) Polyclonal antibody (Proteintech, Rosemont, IL, USA), EIF2S1 polyclonal antibody (Proteintech, Rosemont, IL, USA), alpha tubulin monoclonal antibody (Proteintech, Rosemont, IL, USA)/G3bp1 (E8N8F) rabbit mAb, then HRP-conjugated affinipure goat anti-mouse IgG or HRP-conjugated affinipure goat anti-rabbit IgG (Proteintech, Rosemont) were labeled for color development. The blots were visualized using an ECL kit.
6
Quantitative Protein Expression Analysis
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Total protein from ESCC cells was extracted using RIPA lysis buffer (Proteintech Group, Wuhan, China) that was supplemented with the protease inhibitor PMSF and was quantified by Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s protocols. Equal quantities of proteins were separated by 10% SDS-PAGE and were transferred onto PVDF membranes. After blocking in 5% non-fat milk for 1 h, the membrane was then incubated with E2F1 primary antibody (ab179445; Abcam) at 4 °C overnight. Protein bands were detected using a horseradish-peroxidase (HRP)-conjugated IgG secondary antibody (Proteintech Group), and images were captured with the BioSpectrum imaging system (UVP, USA). GAPDH (60004-1-lg; Proteintech Group) was used as loading control.
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