Rabbit anti scf, by Abcam - Product details

1

Bone Marrow Protein Extraction and Western Blot

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Bone marrow was flushed out of the bone and then dissociated in 66% Trichoracetic acid (TCA) in water. Extracts were incubated on ice for at least 15 min and centrifuged at 16,100 × g at 4°C for 10 min. Precipitates were washed in acetone twice and the dried pellets were solubilized in 9M urea, 2% TritonX-100, and 1% DTT. Samples were separated on 4–12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femtochemiluminescence kit (Thermo Scientific). Primary antibodies used: rabbit-anti-SCF (Abcam, catalogue number ab64677, 1:1000) and mouse-anti-Actin (Santa Cruz, clone AC-15, 1:20,000).

Zhou B.O., Yu H., Yue R., Zhao Z., Rios J.J., Naveiras O, & Morrison S.J. (2017). Bone marrow adipocytes promote the regeneration of stem cells and hematopoiesis by secreting SCF. Nature cell biology, 19(8), 891-903.

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2

Protein Expression Analysis in HUVECs

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The pretreated HUVECs were washed by pre-cooling PBS three times, and the proteins were extracted by RIPA lysis containing 1% PMSF (Solarbio, Beijing, China). The concentration of proteins was measured according to the BCA protein assay kit (Solarbio, Beijing, China). The proteins were boiled for 5 min in SDS-PAGE loading buffer, and 30 μg of proteins was loaded to each lane and separated by 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF; Millipore, Billerica, MA, USA) membranes. The membranes were blocked by 5% non-fat milk for 1 h and then immersed in primary antibodies overnight at 4 °C; the primary antibodies are as follows: rabbit anti-GAPDH (1:10000; Proteintech, Chicago, IN, USA), rabbit anti-PLGF (1:1000; Abcam, Cambridge, UK), rabbit anti-SCF (1:10000; Abcam), and rabbit anti-VEGFR2 (1:1000; Cell Signaling Technology, USA). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000; Proteintech) for 1 h. Chemiluminescence reagents (Millipore) were used for the development of band visualization. ImageJ software was used to analyzed the protein expression levels.

Jin S., Yang C., Huang J., Liu L., Zhang Y., Li S., Zhang L., Sun Q, & Yang P. (2020). Conditioned medium derived from FGF-2-modified GMSCs enhances migration and angiogenesis of human umbilical vein endothelial cells. Stem Cell Research & Therapy, 11, 68.

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3

Splenic Endothelial Cell Protein Extraction and Analysis

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Approximately 30,000 CD45⁻Ter119⁻VE-cadherin⁺ splenic endothelial cells were flow cytometrically sorted into 50 μl of 66% Trichoracetic acid (TCA) in water. Extracts were incubated on ice for at least 15 min and centrifuged at 16,100 × g at 4°C for 10 min. Precipitates were washed in acetone twice and the dried pellets were solubilized in 9M urea, 2% TritonX-100, and 1% DTT. Samples were separated on 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femtochemiluminescence kit (Thermo Scientific). Primary antibodies used: rabbit-anti-SCF (Abcam, 1:1000) and mouse-anti-Actin (Santa Cruz, clone AC-15, 1:20,000).

Inra C.N., Zhou B.O., Acar M., Murphy M.M., Richardson J., Zhao Z, & Morrison S.J. (2015). A perisinusoidal niche for extramedullary haematopoiesis in the spleen. Nature, 527(7579), 466-471.

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4

Bone Marrow Protein Extraction and Western Blot

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Bone marrow was flushed out of the bone and then dissociated in 66% Trichoracetic acid (TCA) in water. Extracts were incubated on ice for at least 15 min and centrifuged at 16,100 × g at 4°C for 10 min. Precipitates were washed in acetone twice and the dried pellets were solubilized in 9M urea, 2% TritonX-100, and 1% DTT. Samples were separated on 4–12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femtochemiluminescence kit (Thermo Scientific). Primary antibodies used: rabbit-anti-SCF (Abcam, catalogue number ab64677, 1:1000) and mouse-anti-Actin (Santa Cruz, clone AC-15, 1:20,000).

Zhou B.O., Yu H., Yue R., Zhao Z., Rios J.J., Naveiras O, & Morrison S.J. (2017). Bone marrow adipocytes promote the regeneration of stem cells and hematopoiesis by secreting SCF. Nature cell biology, 19(8), 891-903.

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5

Splenic Endothelial Cell Protein Extraction and Analysis

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Approximately 30,000 CD45⁻Ter119⁻VE-cadherin⁺ splenic endothelial cells were flow cytometrically sorted into 50 μl of 66% Trichoracetic acid (TCA) in water. Extracts were incubated on ice for at least 15 min and centrifuged at 16,100 × g at 4°C for 10 min. Precipitates were washed in acetone twice and the dried pellets were solubilized in 9M urea, 2% TritonX-100, and 1% DTT. Samples were separated on 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femtochemiluminescence kit (Thermo Scientific). Primary antibodies used: rabbit-anti-SCF (Abcam, 1:1000) and mouse-anti-Actin (Santa Cruz, clone AC-15, 1:20,000).

Inra C.N., Zhou B.O., Acar M., Murphy M.M., Richardson J., Zhao Z, & Morrison S.J. (2015). A perisinusoidal niche for extramedullary haematopoiesis in the spleen. Nature, 527(7579), 466-471.

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6

SCF and c-kit Expression in Prostate Cancer

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Frozen prostate cancer sections were obtained and stained with rabbit anti-SCF (1:100; Abcam) and mouse anti-CD117/c-kit (1:400; Cell Signaling) antibodies after fixation in 4% paraformaldehyde. After incubation with primary antibodies, samples were washed and exposed goat anti-rabbit Alexa Fluor488 and anti-mouse Alexa Fluor568 (Invitrogen) secondary antibodies. The slides were mounted with medium containing DAPI (Dako) and images were taken by a TCS-SP (Leica) microscope. For quantification, the images were analyzed with ImagePro software (Media Cybernetics).

Kerr B.A., Miocinovic R., Smith A.K., West X.Z., Watts K.E., Alzayed A.W., Klink J.C., Mir M.C., Sturey T., Hansel D.E., Heston W.D., Stephenson A.J., Klein E.A, & Byzova T.V. (2014). CD117+ cells in the circulation are predictive of advanced prostate cancer. Oncotarget, 6(3), 1889-1897.

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Rabbit anti scf

Lab products found in correlation

6 protocols using rabbit anti scf

Bone Marrow Protein Extraction and Western Blot

Protein Expression Analysis in HUVECs

Splenic Endothelial Cell Protein Extraction and Analysis

Bone Marrow Protein Extraction and Western Blot

Splenic Endothelial Cell Protein Extraction and Analysis

SCF and c-kit Expression in Prostate Cancer

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