Proteins were extracted with RIPA buffer, uploaded on 10% SDS-PAGE gel, and transferred to PVDF membrane. Membrane was blocked with 5% non-fat milk for 2 h, and then incubated with antibodies (Abcam, Cambridge, CA, USA), including anti-SLC1A5 (1:1,000, ab237704), anti-Bcl-2 (1:1,000, ab32124), anti-Bax (1:1,000, ab32503), anti-YY1 (1:1,000, ab109228), anti-Nanog (1:200, ab21624), anti-Sox2 (1:1,000, ab97959), anti-GAPDH (1:2,500, ab9485), and secondary antibody (1:50,000, ab205718). ECL reagent (Beyotime) was then used for visualizing protein bands, and gray value was analyzed by Image J software.
Ab97959
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Germany
Ab97959 is a Protein A Sepharose resin. It is designed for the purification of antibodies and other Fc-containing proteins from cell culture supernatants, ascites fluid, and other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab19857, by Abcam (16 mentions)
Ab18976, by Abcam (15 mentions)
Ab109250, by Abcam (10 mentions)
Ab80892, by Abcam (9 mentions)
Ab15580, by Abcam (8 mentions)
Ab21624, by Abcam (7 mentions)
Ab8245, by Abcam (7 mentions)
Ab9485, by Abcam (6 mentions)
Ab205718, by Abcam (5 mentions)
Ab5392, by Abcam (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Mab377, by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Ab19898, by Abcam (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Operetta, by PerkinElmer (4 mentions)
Ab181557, by Abcam (4 mentions)
Ab18465, by Abcam (4 mentions)
155 protocols using ab97959
1
Protein Expression Analysis in Stem Cells
2
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tumor tissues were lysed with RIPA Lysis buffer (Beyotime, Shanghai, China), and protein concentrations were determined by the BCA kit (Beyotime, Shanghai, China). Each sample was separated by 8-12% SDS-PAGE, blocked with 5% BSA (Albumin from bovine serum) for 2 h at room temperature, and incubated with primary antibodies to β-tubulin, Sox2 (#14962, 1:100, CST, MA, USA; sc-365964, 1:100, Santa Cruz, TX, USA; ab97959, 1:1000, Abcam, Cambridge, UK), AhR (ab2769, 1:500, Abcam, Cambridge, UK), STAT3 (#12640, 1:1000, CST, MA, USA), Y-STAT3 (ab76315, 1:2000, Abcam, Cambridge, UK), S-STAT3 (ab86430, 1:250, Abcam, Cambridge, UK), PCNA (#2586, 1:2000, CST, MA, USA), IDO1 (#51851, 1:500, CST, MA, USA), p53 (#2524, 1:1000, CST, MA, USA), p27 (#3686, 1:1000, CST, MA, USA), and p21 (sc-397, 1:200, Santa Cruz, TX, USA) overnight at 4 oC. Primary antibodies were detected with a goat anti-rabbit IgG-HRP (sc-2004, 1:2000, Santa Cruz, TX, USA) or anti-mouse IgG-HRP (sc-2005, 1:2000, Santa Cruz, TX, USA). The blots were developed using Super Signal West Pico chemiluminescent substrate (Millipore, Billerica, MA, USA).
3
Immunohistochemical Staining of SOX2, E-cadherin, and Vimentin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining was performed using the following antibodies: Rabbit monoclonal anti-SOX2 antibody (ab97959, Abcam, 1:1000 dilution), rabbit monoclonal anti-E-cadherin antibody (ab227639, Abcam, 1:500 dilution), and rabbit monoclonal anti-vimentin antibody (ab92547, Abcam, 1:500 dilution).
Formalin-fixed samples were embedded in paraffin and then subjected to deparaffinization/ hydration and incubation at 58 to 65 °C for 1 h. On the day before the assays, sections were put into a 37 °C oven overnight to soften the wax layer. After dewaxing and dehydration, the sections were added to a boiling EDTA repair solution (pH 9.0) for 15 min, cooled at room temperature for 30 min, and then endogenous peroxidase was added for 20 min. The sections were then rinsed three times with phosphate-buffered saline, a goat anti-rabbit secondary antibody (PV-6001) was added, and the sections were then maintained at 37 °C for 1 h. Finally, DAB was added for staining. Each experiment included positive control sections and negative control sections, in which PBS replaced the primary antibody.
Formalin-fixed samples were embedded in paraffin and then subjected to deparaffinization/ hydration and incubation at 58 to 65 °C for 1 h. On the day before the assays, sections were put into a 37 °C oven overnight to soften the wax layer. After dewaxing and dehydration, the sections were added to a boiling EDTA repair solution (pH 9.0) for 15 min, cooled at room temperature for 30 min, and then endogenous peroxidase was added for 20 min. The sections were then rinsed three times with phosphate-buffered saline, a goat anti-rabbit secondary antibody (PV-6001) was added, and the sections were then maintained at 37 °C for 1 h. Finally, DAB was added for staining. Each experiment included positive control sections and negative control sections, in which PBS replaced the primary antibody.
4
Immunohistochemical Analysis of Tumor Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors generated in mice were dissected, fixed in 4% formalin for 48 h and embedded in paraffin. Four-micrometer thick sections were deparaffinized, rehydrated and heated for 10 min in citrate buffer for antigen retrieval. Endogenous peroxidase was blocked with 5% hydrogen peroxide in methanol for 15 min. After incubation with blocking solution, sections were incubated with the respective primary antibody, anti-SOX2 (ab97959 Abcam, Cambridge, UK), anti-Ki67 (ab15580, Abcam, Cambridge, UK), anti-p21CIP1 (sc-397 Santa Cruz, Dallas, TX, USA) and anti-p27KIP1 (sc-1641 Santa Cruz, Dallas, TX, USA), at 37 °C for 2 h. The sections were then washed and incubated with MACH 3 Rabbit Probe and MACH 3 Rabbit HRP-Polymer (M3R531, Biocare Medical, Pacheco, CA, USA). Color was developed with 3,3′-Diaminobenzidine (DAB) and nuclei were counterstained with hematoxylin.
5
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected in Cell Extraction Buffer (Invitrogen, FNN0011) with protease inhibitor (Roche, 04693116001) and PMSF (Sigma, P7626). Proteins were separated on 10% or 12% SDS-PAGE gels before transfer to Millipore Immobilon-P PVDF Membrane (Merck Millipore, IPVH00010). Membranes were probed with pAKT(S473) (Cell Signaling, 3787, 1:3,000), pan-AKT (Cell Signaling, 4691, 1:1,000), dpERK1/2 (Cell Signaling, 4370, 1:300), ERK1/2 (Cell Signaling, 4695, 1:300), SPRY2 (Abcam, Ab50317, 1:300), SOX2 (Abcam, Ab97959, 1:3,000), Histone H3 (Abcam, Ab39655, 1:10,000), and EGFR (Millipore, 04-290, 1:300). Detection was performed with horseradish peroxidase-conjugated secondaries (Abcam, ab97051, ab97023, 1:10,000) and enhanced chemiluminescence (Thermo Scientific, PI-32109). Quantitation is based on analysis of protein from three biological replicates separated on the same polyacrylamide gel. Band intensity was analyzed in Fiji normalized to loading control.
6
Western Blot Analysis of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using a Protein Extraction kit (P0027; Beyotime Institute of Biotechnology, Beijing, China). Whole cell protein concentrations were measured using BCA reagent (P0010S; Beyotime Institute of Biotechnology). Lysates (25 µg total protein) were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated in blocking buffer (3% bovine serum albumin; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Subsequently, the membranes were incubated at 4°C overnight with the following primary antibodies: GAPDH (ab9485; 1:2,500), Oct-4 (ab19857; 1:1,000), Nanog (ab109250; 1:1,000) and Sox2 (ab97959; 1:1,000; all purchased from Abcam, Cambridge, MA, USA). Goat anti-rabbit secondary antibody conjugated with peroxidase (BS13278; 1:5,000; Bioworld Technology, Inc., St Louis Park, MN, USA) was used as the secondary detection antibody at 25°C for 1 h. The immunoblots were then detected using an enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.) on the ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Images were analyzed using Image Lab software 3.0 (Bio-Rad Laboratories, Inc.).
7
Quantification of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA (Cell signalling) and protease inhibitors (Roche) as per manufacturer instructions. Total protein was measured using the bicinchoninic acid (BCA) method (Pierce Biotechnology). In total, 50 mg cell lysate was separated using 7.5% SDS–PAGE gels and transferred to PVDF membranes by electro-blotting. Membranes were blocked in 5% (w/v) milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). Blots were then incubated at 4 °C overnight with primary antibodies as indicated, washed in TBST and subsequently probed with secondary antibodies for 1 h at room temperature. ECL solution was then added to the membrane and analysed. Antibodies used were, anti-BCL11A (ab191401, Abcam, 1:3000), anti-SOX2 (ab97959, Abcam, 1:2000) and anti-TUBULIN (ab7291, Abcam, 1:10000). All the original western blot images can be found in Supplementary Figures 14 and 15 .
8
Immunostaining of Human iPSCs and EBs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunostaining, human iPSCs and EBs were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature, washed with PBS, and permeabilized in 0.3% Triton X-100 in PBS for 10 min at room temperature. Primary antibodies used were anti-Oct4 (ab18976, Abcam, Cambridge, UK), anti-Nanog (ab62734, Abcam), anti-SOX2 (ab97959, Abcam), anti-TRA-1-60 (ab16288, Abcam), anti-Nestin (ab22035, Abcam), anti-α-SMA (ab5694, Abcam), anti-Brachyury (AF2085, R&D Systems, Minneapolis, MN, USA), and anti-GATA4 (ab134057, Abcam). After primary antibody incubation, samples were washed with PBS and incubated with secondary Alexa Fluor 488-conjugated antibodies (Life Technologies), diluted 1:2,000. Samples were also counterstained with DAPI (200 μg/mL). Slides were observed using an Olympus BX61 research microscope equipped with a cooled CCD camera. Images were captured and analyzed with Applied Imaging software CytoVision.
9
Immunofluorescence Analysis of Stemness Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tumorspheres were centrifuged at 200 × g for 5 min to remove media, washed with PBS and collected in EP tubes at room temperature. Next, the tumorspheres were fixed in 4% paraformaldehyde in the EP tubes. Rabbit anti-human primary antibodies, including Sox2 (ab97959; 1:1,000), Oct4 (ab19857; 1:200) and Nanog (ab109250; 1:200; all from Abcam), were added and incubated overnight at 4°C using a shaking table. Subsequent to washing the tumorspheres three times with PBS, goat anti-rabbit secondary antibodies conjugated with Cy3 (P0183; Beyotime Institute of Biotechnology; 1:1,500) were added, and the tumorspheres were incubated at room temperature for 1 h. DAPI was used as the nuclear stain (blue). Images were obtained using fluorescence microscopy.
10
Immunohistochemical Analysis of Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mice were perfused and fixed with 4% paraformaldehyde in PBS overnight at 4 °C. The brains, spinal cord and other organs were removed and sectioned at 30 μm thickness with a Leica CM1950 cryostat (Leica). Slices were permeabilized with blocking solution containing 0.1% Triton X-100 and 1% BSA in PBS for 1 h at room temperature and then incubated with primary antibody overnight at 4 °C. The following day, after the slices were washed with PBS, they were incubated with fluorescently conjugated secondary antibodies (Invitrogen) and DAPI (Sigma) for 1 h at room temperature. The slices were then washed and mounted in Dako (s3023) mounting medium. Images of stained slices were acquired by a confocal microscope (Olympus FV1000) with various objective lenses. The images were analysed with ImageJ software. The following primary antibodies were used: mouse anti-flavivirus Envelope protein (AbFLAVENV-4G2), anti-Sox2 (Abcam/ab97959), anti-Tbr1 (Abcam/ab31940), anti-NeuN (Merk/ABN 78), anti-p-Histone H3 (Ser 10, Santa Cruz/sc-8656-R), anti-cleaved caspase-3 (Cell Signalling/Asp175), anti-Ki67 (Abcam/ab15580), and anti-doublecortin (Abcam/ab77450).
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!