Proteins were extracted by solubilizing the cells in boiling SDS buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1% SDS). Samples were boiled for 5 min at 95 °C and sonicated for 10 s. Extracts were clarified by centrifugation, normalized with the BCA Protein Assay Reagent kit (Thermo). Equal amounts of proteins (20 μg) were loaded in each lane. Proteins were separated by PAGE and transferred to nitrocellulose sheets. Western blot detection was performed with enhanced chemiluminescence system (GE Healthcare) and peroxidase-conjugated secondary antibodies (Amersham). The following primary antibodies were used for western blotting: anti-beta2 Microglobulin [EP2978Y] (ab75853, Abcam), anti-MLH1 (ab92312, Abcam), anti-MSH2 (ab70270, Abcam), anti-MSH6 [EPR3945] (ab92471, Abcam), anti-MSH3 PA527864, Invitrogen, anti-PMS2 EPR3947 (Cell Marque Corporation, USA), anti-actin (I-19) (sc1616, Santa Cruz), and anti-HSP 90α/β (H-114, sc-7947, Santa Cruz). Images were acquired with Chemidoc (Biorad), and western blot band intensity was analyzed using Image Lab software (Biorad).
Ab75853
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab75853 is a Recombinant Protein A Conjugate. It is a laboratory reagent used for protein purification and immunodetection.
Automatically generated - may contain errors
Lab products found in correlation
Amersham hyperfilm ecl, by GE Healthcare (2 mentions)
Nupage lds loading buffer, by Thermo Fisher Scientific (2 mentions)
Na934, by GE Healthcare (2 mentions)
Gradient sds polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by GE Healthcare (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Ab70328, by Abcam (2 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ab92312, by Abcam (1 mentions)
Ab92471, by Abcam (1 mentions)
Ab70270, by Abcam (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
Bca protein assay reagent kit, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Anti pkcθ clone 27 pkcθ, by BD (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab108349, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Maclin Biochemical Technology (1 mentions)
12 protocols using ab75853
1
Protein Extraction and Western Blot Analysis
2
Protein Extraction and Western Blotting
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Proteins were extracted in 50 mM Tris, 120 mM NaCl, 1 mM EDTA with 1% IGEPAL CA-630 and protease and phosphatase inhibitor cocktails (Thermo) before SDS-PAGE and western blotting. The antibodies used in this study were anti-PKCθ clone 27/PKCθ (BD Bioscience), anti-PKCθ pT538 ployclonal (Cell Signaling Technologies #9377) , anti-c-Jun Rabbit monoclonal (Cell Signaling Technologies #9165), anti-c-fos Rabbit monoclonal (Cell Signaling Technologies #2250), anti-fosB Rabbit monoclonal (Cell Signaling Technologies #2251), anti-JunB Rabbit polyclonal (Santa Cruz Biotehnology #sc-46X) anti-JunD Rabbit polyclonal (Santa Cruz Biotehnology #sc-74X) and anti-beta-2-microglobulin (Abcam #AB75853).
3
Quantifying Hippocampal Protein Levels
4
Chondrocyte Senescence and Regeneration
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Anhydrous copper chloride (CuCl2), 3,3’,5,5’-tetramethylbenzidine (TMB), bovine serum albumin (BSA), and sodium sulfide nonahydrate (Na2S·9H2O) were from Macklin Biochemical (Shanghai, China). Doxorubicin (Dox), N-(3-Dimethylaminopropyl)-N’-ethylcabodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were from Sigma-Aldrich (USA). Antibodies against p16ink4a (polyclonal, ab108349), B2M (monoclonal, ab75853), high mobility group protein 1 (HMGB-1) (ab228624), matrix metalloprotein 13 (MMP-13) (ab39012), and type II collagen (Col-2) (ab34712) were from Abcam (UK). The cellular senescence β-galactosidase staining kit and cell counting kit (CCK-8) were from Beyotime Biotechnology (Shanghai, China). FITC-xtra and MitoROS™ 580 were from ATT Bioquest (USA). The Evo M-MLV Reverse Transcription Reagent and SYBR Green Pro Taq HS qPCR Kit were from Accurate Biology (China). The chondrogenic differentiation kit was from Cyagen Biosciences (USA).
5
Immunohistochemical Analysis of Tissue Samples
6
Glycomic Profiling of Bladder Cancer
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Protein extracts and CD44 immunoprecipitates from 5637 and T24 wild type, glycoengineered cells and bladder tumors were separated in 4-20% precast polyacrylamide gels (Bio-Rad) and transferred onto a nitrocellulose membrane (GE Healthcare Life Sciences). STn and Tn expressions were evaluated using the anti-tag-72 antibody [B72.3 + CC49] (1 μg/mL, ab199002, Abcam; Table S2 ) and biotinylated Vicia Villosa lectin (VVA lectin, 1:1000, Vector Laboratories; Table S2 ), respectively. CD44 expression was screened with the anti-CD44 antibody (1:5000, ab157107, Abcam; Tables S2 ). Proteins were blotted with the primary antibody or lectin during 1 h at RT. The peroxidase affiniPure goat anti-mouse IgG (H+L) polyclonal antibody (1:90,000, ImmunoResearch) was used as a secondary antibody for anti-tag-72 antibody detection, and the goat anti-rabbit IgG (H+L) HPR conjugate antibody (1:60,000; Thermo Fisher Scientific) was used for anti-CD44 antibody detection, both incubated for 30 min at RT. The VECTASTAIN® Elite ABC-HRP Reagent (1:10; Vector Laboratories) was used for 15 min at RT for analysis of Tn expression. Detection of B2M with the recombinant anti-B2M antibody [EP2978Y] (ab75853, Abcam; Table S2 ) followed by incubation with goat anti-rabbit IgG (H+L) HPR conjugate antibody (1:60,000; 30 min at RT) was performed as loading control.
7
Quantifying Hippocampal Protein Levels
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Mouse hippocampi were dissected after perfusion of animals, snap frozen and lysed in RIPA lysis buffer (500 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Na deoxycholate, 1% NP40, 0.1% SDS, and complete protease inhibitors; Roche). Tissue lysates were mixed with 4× NuPage LDS loading buffer (Invitrogen) and loaded on a 4–12% SDS polyacrylamide gradient gel (Invitrogen) and subsequently transferred onto a nitrocellulose membrane. The blots were blocked in 5% milk in Tris-Buffered Saline with Tween (TBST) and incubated with rabbit anti-actin (1:5000, Sigma; A5060) and rabbit anti-B2M (1:2500, Abcam; ab75853; clone: EP2978Y). Horseradish peroxidase-conjugated secondary antibodies (1:5000, GE Healthcare; NA934) and an ECL kit (GE Healthcare/Amersham Pharmacia Biotech) were used to detect protein signals. Multiple exposures were taken to select images within the dynamic range of the film (GE Healthcare Amersham HyperfilmTM ECL). Selected films were scanned (300 dpi) and quantified using ImageJ software (Version 1.46k). Actin bands were used for normalization.
8
Immunoblotting of Frozen Hippocampi
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Frozen hippocampi were pulverized in lysis buffer (20 mM Tris-HCl, pH 7.65, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton) supplemented with 1× protease and phosphatase inhibitor cocktail (78443; Thermo Fisher Scientific). Lysates were clarified by centrifugation, and protein concentration was determined using a Bradford assay. Clarified samples were heated at 65°C in Laemmli buffer for 5 min. Proteins were run on an SDS-PAGE gel and transferred to nitrocellulose membrane (1620112; Bio-Rad). Membranes were blocked with TBST-5% BSA for 1 h, incubated with primary antibody overnight at 4°C, and probed with HRP-conjugated antibodies for 1 h at room temperature. The following antibodies were used in these studies: anti-TIMP2 (1:1,000; D18B7; Cell Signaling), anti–α-tubulin (1:5,000; T6199; Sigma-Aldrich), anti-b2 microglobulin (1:1,000; ab75853; Abcam), antitransferrin (1:1,000; ab82411; Abcam), anti-Gpr158 (1:1,000; ABIN486340; Antibodies-online), anti-Na,K ATPase (1:1,000; 3010S; Cell Signaling), anti-SR2C (1:1,000; sc-17797; Santa Cruz), anti-Gaq (1:1,000; D5V1B; Cell Signaling), anti-BDNF (1:100; sc-65514; Santa Cruz). Band intensities were quantified using ImageJ software. Dashed lines separating two bands indicate that these bands are on the same membrane but are not adjacent.
9
Western Blot Analysis of Protein Expression
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Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. The samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated with primary antibodies overnight. Then, the membranes were incubated with the secondary antibodies for 1 h at room temperature, and the bands were detected using an enhanced chemiluminescence kit. Antibodies against MARC2 (ab224097), PPARA (ab126285), HLA-C (ab126722), and B2M (ab75853) were obtained from Abcam.
10
Immunophenotyping of Murine Cell Populations
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Monoclonal antibodies specific for CD16 and CD32 (Biolegend, 101302; 1:100) were used for blockade of Fc receptors before staining. The antibodies used for cell surface labeling were AF647 anti-mouse H-2Kb/H-2Db (Biolegend, 114612; 1:100), APC anti-mouse Thy1.1 (Biolegend, 202526; 1:100), and PE anti-mouse NK1.1 (Biolegend, 108707; 1:100). Intracellular staining of unlabeled B2M (Abcam, ab75853; 1:100) following staining with a secondary APC anti-rabbit IgG (R&D systems, F0111) was performed using a staining buffer set (eBioscience) according to the manufacturer’s instructions. Samples were analyzed by CytoFLEX flow cytometer (Beckman Coulter).
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