Lung tissues were homogenized and protein was isolated using the RIPA lysis solution (P0013B, Beyotime, Shanghai, China). Protein concentration was determined with a Bradford protein assay kit (P0009, Beyotime). A total of 15–30 µg protein per lane was separated on 10% SDS polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). Following blocking in TBST (TBS with 0.1% Tween-20) for 1 h, the membrane was incubated with anti-S1PR1 (1:500; A12935, ABclonal, Wuhan, China), anti-nuclear factor kappa-B (NF-κB) ligand p65 (1:1,000; A19653, ABclonal), anti-phospho-inhibitor of NF-κB (p-IκB)-α (Ser32/36; 1:500; AF2002, Affinity Biosciences, Changzhou, China), anti-IκB-α (1:1,000; AF5002, Affinity), anti-p-IκB kinase (IKK; Ser180/Ser181; 1:500; AF3013, Affinity, China), anti-IKK (1:1,000; AF6014, Affinity) and anti-Histone H3 (1:500; 17168-1-AP, Proteintech, Wuhan, China), and anti-β-actin (1:2,000; 60008-1-Ig, Proteintech) antibodies at 4°C overnight. Membranes were incubated with the secondary antibodies at 37°C for 40 min. The relative intensity of proteins was visualized using electrochemiluminescence reagents (Shanghai 7sea biotech Co., Ltd.) and quantified using Gel-Pro Analyzer 4.0 (Media Cybernetics, Rockville, MD, USA).
Af2002
Manufactured by Affinity Biosciences
Sourced in United States, China
The AF2002 is a benchtop centrifuge designed for general laboratory use. It can accommodate a range of rotor sizes and tube capacities to handle a variety of sample types and volumes. The centrifuge features digital speed and time controls for precise operation.
Automatically generated - may contain errors
Lab products found in correlation
Af5002, by Affinity Biosciences (6 mentions)
Af5006, by Affinity Biosciences (5 mentions)
Af2006, by Affinity Biosciences (4 mentions)
Af3013, by Affinity Biosciences (3 mentions)
Af7021, by Affinity Biosciences (2 mentions)
Af6009, by Affinity Biosciences (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Necrostatin 1, by Selleck Chemicals (2 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
A19653, by ABclonal (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
Ripa lysis solution, by Beyotime (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Ab73400, by Abcam (1 mentions)
Ab214821, by Abcam (1 mentions)
Ab32518, by Abcam (1 mentions)
Ab32539, by Abcam (1 mentions)
Ab2796, by Abcam (1 mentions)
Ab32041, by Abcam (1 mentions)
11 protocols using af2002
1
Quantification of Protein Expression in Lung Tissues
2
Western Blot Analysis of Osteoblast Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins of MC3T3-E1 cells or BMSCs were extracted and lysed with RIPA buffer (Beyotime, China), and protein concentration was detected using a BCA protein assay kit (Solarbio, China). Then, a 20 μg total protein sample was loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation and transferred to a PVDF membrane. After being blocked with 5% skimmed milk, the membrane was incubated at 37°C with the primary antibodies against caspase-3 (ab13847, Abcam), caspase-9 (ab32539, Abcam), SIRT1 (ab110304, Abcam), BMP-2 (ab214821, Abcam), RANKL (ab45039, Abcam), OPG (ab73400, Abcam), IκBα (ab32518, Abcam), p-IκBα (AF2002, Affinity), p65 (ab207297, Abcam), p-p65 (AF2006, Affinity), IKKα (ab32041, Abcam), p-IKKα (AF3013, Affinity), NFATc1 (ab2796, Abcam), and cathepsin K (ab19027, Abcam) at 4°C overnight. After that, the membrane was further incubated with the secondary antibody at room temperature for 1 h. Then, the enhanced chemiluminescence (ECL) reagent (Solarbio, China) was used to visualize the protein bands, and the relative band density was semiquantified with the ImageJ software.
3
Lung Tissue Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The inferior lobe of the lung tissues was homogenized in RIPA lysis buffer (Thermo Fisher Scientific, Inc.). After that, 30 μg protein was separated in 10% SDS-PAGE. Proteins were then transferred to PVDF membranes (Millipore). Then, the PVDF membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated with primary antibodies against p-P65 (1 : 1000, AF2006, Affinity); P65 (1 : 1000, AF5006, Affinity); p-IKBα (1 : 1000, AF2002, Affinity); IKBα (1 : 1000, AF5002, Affinity); NLRP3 (1 : 1000, DF7438, Affinity), ASC (1 : 2000, sc-514414, Santa Cruz Biotechnology, INC), Caspase-1 p20 (1 : 2000, AF4005, Affinity), Pro-GSDMD (1 : 1000, AF4012, Affinity), GSDMD p30 (1 : 1000, DF12275, Affinity), IL-18 (1 : 1000, DF6252, Affinity), IL-1β (1 : 1000, AF5103, Affinity), and β-actin (1 : 5000, AF7018, Affinity) at 4°C overnight. Then, the membranes were then incubated with the appropriate secondary antibodies at room temperature for 2 h. β-actin was selected as the loading control. Finally, the protein bands were caught by using a Chemiluminescence image analysis system (Tanon, Shanghai, China) and analyzed with the Image J software (National Institutes of Health, USA).
4
Microglia Polarization Evaluation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CMCS (MW 20 − 30 kDa, degree of carboxylation ∼80%, Beijing Solarbio Science & Technology Co., Ltd., CHN), TA (J&K Scientific Ltd., CHN), Cy5 (RuixiBio, CHN), phosphate buffered saline (PBS, Solarbio, CHN), normal saline (0.9%, China Resources Double-crane Pharmaceutical Co., Ltd., CHN), paraformaldehyde (PFA, 4%, Solarbio, CHN), sodium pentobarbital (Merck, Germany), and rose bengal (Sigma-Aldrich, Germany) were used.
Primary antibodies for IF staining:
Rabbit anti-IBA-1 (ab178846, Abcam, UK), mouse anti-iNOS (MA5-17,139, Invitrogen), goat anti-CD206 (AF2535, R&D), mouse anti-PSD95 (ab2723, Abcam, UK), and rabbit anti-Vglut1 (GTX133148, GeneTex, USA) were used.
Primary antibodies for WB:
Rabbit anti-CD206 (24,595, CST, USA), rabbit anti-iNOS (GB113965, Solarbio, CHN), mouse anti-p65 (GB12142, Solarbio, CHN), rabbit anti-p-p65 (GB113882, Solarbio, CHN), rabbit anti-IĸBα (GB111509, Solarbio, CHN), rabbit anti-p-IĸBα (AF2002, AFFinity, USA), rabbit anti-Synaptophysin (GB11814, Solarbio, CHN), rabbit anti-PSD95 (GB11277, Solarbio, CHN), rabbit anti-CD16 (80,006, CST, USA), rabbit anti-IL-1β (GB11113, Solarbio, CHN), rabbit anti-TGF-β (GB111876, Solarbio, CHN), rabbit anti-TLR4 (GB11519, Solarbio, CHN), rabbit anti-LaminB1 (GB111802, Solarbio, CHN), and mouse anti-GAPDH (GB15002, Solarbio, CHN) were used.
Primary antibodies for IF staining:
Rabbit anti-IBA-1 (ab178846, Abcam, UK), mouse anti-iNOS (MA5-17,139, Invitrogen), goat anti-CD206 (AF2535, R&D), mouse anti-PSD95 (ab2723, Abcam, UK), and rabbit anti-Vglut1 (GTX133148, GeneTex, USA) were used.
Primary antibodies for WB:
Rabbit anti-CD206 (24,595, CST, USA), rabbit anti-iNOS (GB113965, Solarbio, CHN), mouse anti-p65 (GB12142, Solarbio, CHN), rabbit anti-p-p65 (GB113882, Solarbio, CHN), rabbit anti-IĸBα (GB111509, Solarbio, CHN), rabbit anti-p-IĸBα (AF2002, AFFinity, USA), rabbit anti-Synaptophysin (GB11814, Solarbio, CHN), rabbit anti-PSD95 (GB11277, Solarbio, CHN), rabbit anti-CD16 (80,006, CST, USA), rabbit anti-IL-1β (GB11113, Solarbio, CHN), rabbit anti-TGF-β (GB111876, Solarbio, CHN), rabbit anti-TLR4 (GB11519, Solarbio, CHN), rabbit anti-LaminB1 (GB111802, Solarbio, CHN), and mouse anti-GAPDH (GB15002, Solarbio, CHN) were used.
5
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment, the cells were harvested and lysed using a cell buffer (WB038, GEFAN, Nanjing, China), and their concentrations were estimated by a BCA kit (GM03, GEFAN). After denaturation, cells were subjected to protein electrophoresis. After transferring to a PVDF membrane (WB041, GEFAN), the membrane was sealed with 5% skim milk. After 2 h, cells were reacted with primary antibodies overnight at 4°C followed by incubation with a goat anti-rabbit secondary antibody IgG H&L (1:3000, S0001, Affinity Biosciences, Cincinnati, OH, USA) for 1 h at 37°C. Finally, a color reagent (E266188, Aladdin, Shanghai, China) was used to visualize the bound antibodies, which were then placed in a chemiluminescent instrument (610020-9Q, Clinx, Shanghai, China). The primary antibodies of the rabbit anti-human Nrf2 (1:2000, AF0639), SOD-1 (1:1500, AF5198), CAT (1:2000, AF7746), HO-1 (1:1500, AF5393), nuclear factor kappa B (NF- κB, 1:1500, AF5006), phospho-IkappaB-alpha (p-IKBα, 1:2000, AF2002), and GAPDH (1:20000, AF7021) were obtained from Affinity Biosciences .
6
Protein Expression Analysis in Kidney Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidney tissues were homogenized and lysed in RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (25 μg) were separated using SDS-PAGE and transferred to PVDF membranes (Beyotime). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti-IRAK2 (1 : 1,000; #DF4782, Affinity, MI, USA), anti-p-NF-κB (1 : 1000; #AF2002, Affinity), anti-NF-κB (1 : 1,000; #AF5006, Affinity), anti-p-IKKβ (1 : 1,000; #AF3010, Affinity), anti-IKKβ (1 : 1,000; #AF6009, Affinity), and anti-GAPDH (1 : 5,000; #AF7021, Affinity). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (1 : 5,000; #S0001, Affinity) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific) and quantified using the ImageJ software (National Institutes of Health, MD, USA).
7
Profiling Inflammatory Signaling in Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total liver tissue homogenates and whole cell lysates were used for Western blotting to determine (i) protein levels of proinflammation cytokines: TNF-α (Proteintech, 60291-1-Ig, 1:1000) and IL-1β (Abcam, ab234437, 1:1000); (ii) the activation state of NF-κB p50/p65 signals including NF-κB p105 (Proteintech, 66992-1-Ig, 1:1000), p-NF-κB p50 (Abclonal, AP0125, 1:1000)/NF-κB p50 (Proteintech, 66992-1-Ig, 1:1000), p-NF-κB p65 (Affinity,AF3388, 1:2000)/NF-κB p65 (Abclonal, A19653, 1:500), and p-IκB (Affinity,AF2002, 1:2000)/IκB (Affinity,AF5002, 1:2000); and (iii) protein levels of Sema7a (Proteintech, 67397-1-Ig, 1:1000) and integrin β1 (Abcam, ab183666, 1:5000). Quantification of Western blotting results was determined by densitometric scanning using ImageJ software. Raw data of Western blotting results were included in Additional file 4 .
8
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenocytes were lysed in fresh extraction buffer (Sigma-Aldrich, St. Louis, MO,
USA) supplemented with a protease inhibitor and phosphatase inhibitor (Gold
Biotechnology, St. Louis, MO, USA). The extracted proteins (20 μg) were
separated on a 15% SDS-polyacrylamide gel and electroblotted onto a
polyvinylidene fluoride membrane. The membrane was blocked using 5% milk
dissolved in TBST for 60 min at 20–25°C and incubated overnight at 4°C with
primary antibodies against p-p38 (AF4001, Affinity Biosciences, Changzhou,
China), p38 (AF6456, Affinity Biosciences) p-p65 (AF2006, Affinity Biosciences),
p65 (AF5006, Affinity Biosciences), p-IκBα (AF2002, Affinity Biosciences) and
IκBα (AF5002, Affinity Biosciences). The membranes were then washed with TBST
and incubated with goat anti-mouse IgG (1:1000; HAF007, R&D Systems, Inc.,
Minneapolis, MN, USA) and goat anti-rabbit IgG (1:1000; HAF008, R&D Systems,
Inc., Minneapolis, MN, USA). Bound antibodies were detected using an
electrochemiluminescence detection system (Amersham Life Science, Arlington
Heights, IL, USA). β-Actin was used as the control and to ensure equal protein
loading.
USA) supplemented with a protease inhibitor and phosphatase inhibitor (Gold
Biotechnology, St. Louis, MO, USA). The extracted proteins (20 μg) were
separated on a 15% SDS-polyacrylamide gel and electroblotted onto a
polyvinylidene fluoride membrane. The membrane was blocked using 5% milk
dissolved in TBST for 60 min at 20–25°C and incubated overnight at 4°C with
primary antibodies against p-p38 (AF4001, Affinity Biosciences, Changzhou,
China), p38 (AF6456, Affinity Biosciences) p-p65 (AF2006, Affinity Biosciences),
p65 (AF5006, Affinity Biosciences), p-IκBα (AF2002, Affinity Biosciences) and
IκBα (AF5002, Affinity Biosciences). The membranes were then washed with TBST
and incubated with goat anti-mouse IgG (1:1000; HAF007, R&D Systems, Inc.,
Minneapolis, MN, USA) and goat anti-rabbit IgG (1:1000; HAF008, R&D Systems,
Inc., Minneapolis, MN, USA). Bound antibodies were detected using an
electrochemiluminescence detection system (Amersham Life Science, Arlington
Heights, IL, USA). β-Actin was used as the control and to ensure equal protein
loading.
9
Investigating Necroptosis Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following commercial antibodies and reagents were used: Necrostatin-1 (S8037, Selleck); Necrostatin 1S (S8641, Selleck); Z-VAD-FMK (S0723, Selleck); TPCA-1(S2824, Selleck); PH-797804 (S2726, Selleck); SP600125 (S1460, Selleck); C-176 (S6575, Selleck); QNZ (S4902, Selleck); RIPK1 (1:1000, 551041, BD Biosciences); RIPK3 (1:1000, NBP1-77299, NOVUS); MLKL (1:2000, AP14272B, Abcepta); pMLKL (1:1000, 37333S, Cell Signaling Technology); Flag (1:1000, 20543-1-AP, Proteintech); caspase3 (1:1000, 19677-1-AP, Proteintech); HMGB1 (1:1000, 10829-1-AP, Proteintech); p65 (1:1000, 66535-1-Ig, Proteintech); GAPDH (1:10,000, 60004-1-Ig, Proteintech); Histone H3 (1:1000, 17168-1-AP, Proteintech);IL6 (1:1000, 66146-1-Ig, Proteintech); CD68 (1:1000, 28058-1-AP, Proteintech); TMEM119 (1:1000, 27585-1-AP, Proteintech); p-p65 (1:200, sc-136548, Santa Cruz); IKK (1:500, AF6009, Affinity); p-IKK (1:500, AF3013, Affinity); IKbα (1:1000, 10268-1-AP, Proteintech); p-IKbα (1:500, AF2002, Affinity); Ccl5 (1:500, AF5151, Affinity); IFNb1 (1:500, DF6471, Affinity); pTau396 (1:1000, 829,001, Biolegend); AT8 (1:1000, MN1020, Thermo); Goat Anti-Mouse-HRP IgG (1:10,000, 115-035-003, Jackson ImmunoResearch); Goat Anti-Rabbit-TRITC IgG (1:200, 111-025-045, Jackson ImmunoResearch); Goat Anti-Rabbit-HRP IgG (1:10,000, 111-005-003, Jackson ImmunoResearch).
10
Protein Expression Analysis of Neuroinflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The BV2 or SH-SY5Y cells or midbrain tissue lysates were subjected to Western blotting to detect NFATc2, STAT1, p-STAT1, IRF9, BAX, BCL-2, p65, p-p65, IκBα, p-IκBα and GAPDH. The following antibodies were used for protein detection by western blotting. NFATc2 (22023–1-AP, 1:1000, Proteintech), STAT1 (66545–1-lg, 1:2000, Proteintech), p-STAT1 (ab109461, 1:1000, Abcam), IRF9 (14167–1-AP, 1:1000, proteintech), BAX (A19684, 1:1000, ABclonal), BCL-2 (26593–1-AP, 1:1000, Proteintech), NF-kB p65 (AF5006, 1:1000, Affinity), Phospho-NF-kB p65(Ser536) (AF2006, 1:1000, Affinity), IKB alpha (AF5002, 1:1000, Affinity), Phospho-IKB alpha (Ser32/Ser36) (AF2002, 1:1000, Affinity), and GAPDH (#5174, 1:1000, Cell Signaling Technology).
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
Revolutionizing how scientists
search and build protocols!