Anti cd44

Manufactured by Proteintech

Sourced in United States, China, United Kingdom

Anti-CD44 is a laboratory antibody product used to detect the CD44 protein. CD44 is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. The Anti-CD44 product can be used in various applications to study CD44 expression and function.

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Lab products found in correlation

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Close spelling variants of this product

Anti-CD44 antibody (3 citations) Anti-CD44 (15675-1-AP) (3 citations) Anti-TM (2 citations) Rabbit anti-CD44 (2 citations) Anti-CD44 polyclonal antibody (2 citations)

23 protocols using anti cd44

Evaluating Cell Invasion and Stemness

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For cell invasion experiments, 2x10 5 cells were seeded into the upper chamber of a Matrigel-coated Boyden chamber in serum-free DMEM. The lower chamber was supplemented with DMEM containing 20% FBS as a chemoattractant. The cells were incubating for 48 h and the chamber was fixed with 10% neutral formalin for >4 h. The cells were dyed with crystal violet (Beyotime). The cells were counted under a microscope (Olympus) and the cell number is expressed as the average number of the cells in each field.
Flow cytometric analysis. The GBC cells were incubated with the primary anti-CD44 (Cat. no. 15675-1-AP; Proteintech, Rosemont, IL, USA) or anti-CD133 (Cat. no. 18470-1-AP; Proteintech) for 30 min at room temperature. The cells were then subjected to flow cytometry using a MoFlo XDP cell sorter from Beckman Coulter (Indianapolis, IN, USA) according to the manufacturer's instructions.
The GBC-SD LKB1 or SGC-996 LKB1 and their control cells were incubated with the primary anti-CD44 (Cat. no. 15675-1-AP; Proteintech) or anti-CD133 (Cat. no. 18470-1-AP; Proteintech) for 30 min at room temperature. Flow cytometric analysis was performed using a MoFlo XDP cell sorter from Beckman Coulter according to the manufacturer's instructions.

Hu M.T., Wang J.H., Yu Y., Liu C., Li B., Cheng Q.B, & Jiang X.Q. (2018). Tumor suppressor LKB1 inhibits the progression of gallbladder carcinoma and predicts the prognosis of patients with this malignancy. International journal of oncology, 53(3).

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Comprehensive Western Blot Protocol

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Western blot was performed as previously described 44 (link). The antibodies included anti-Oct4 (Bioss, Beijing, China, bs-0830R, 1:1000), anti-Nanog (Proteintech, Wuhan, China, 14295-1-AP, 1:1000), anti-CD44 (Proteintech, 15675-1-AP, 1:1000), anti-Sox2 (Proteintech, 11064-1-AP, 1:1000), anti-γ-H2AX (Abcam, Cambridge, UK, ab26350, 1:1000), anti-DLG2 (Affinity Biosciences, Suzhou, China, DF3995, 1:1000), anti-p-Yap1 (CST, Boston MA, USA, 4911S, 1:1000), anti-Yap1 (Proteintech, 13584-1-AP, 1:1000), anti-Bax (CST, B8429, 1:1000), anti-Bcl2 (Proteintech, 60178-1-Ig, 1:1000), anti-β-tubulin (Sigma-Aldrich, T5201, 1:1000), anti-Lamin B1 (Santa, Dallas TX, USA, sc-365962, 1:1000), anti-TEAD1 (ABclonal, Wuhan, China, A13366, 1:1000), anti-β-actin (Santa, sc-8432, 1:4000), CD63 (Proteintech, 25682-1-AP, 1:1000), and TSG101 (Proteintech, 28283-1-AP, 1:1000).

Zou Y., Zhao Z., Wang J., Ma L., Liu Y., Sun L, & Song Y. (2023). Extracellular vesicles carrying miR-6836 derived from resistant tumor cells transfer cisplatin resistance of epithelial ovarian cancer via DLG2-YAP1 signaling pathway. International Journal of Biological Sciences, 19(10), 3099-3114.

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Immunophenotyping of Cultured Cells

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IHC analysis was performed on the cell-block sections from the cultured cells by using the following primary antibodies: anti-CD133 antibody (1:100; Proteintech), anti-CD44 (1:100, Proteintech), anti-c-MYC (1:100; Proteintech), and anti-Ki-67 (MXB, Fuzhou, China).
Immunofluorescence and confocal microscopy were performed as previously reported [19 (link), 20 (link)]. Images were captured by using a Zeiss confocal microscope.

Peng J., Shi S., Yu J., Liu J., Wei H., Song H., Wang S., Li Z., He S., Li L., Zhang H., Yan Z., Zhao R., Liu Y., Liu Y., Li J., Zhang R, & Wang W. (2021). miR-378d suppresses malignant phenotype of ESCC cells through AKT signaling. Cancer Cell International, 21, 702.

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Western Blot Analysis Protocol

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Western blot was performed as previously described [24 (link)]. The primary antibodies used were: anti-CHRNB2 (Abcam, ab41174) for Fig. 5, anti-CHRNB2 (Santa Cruz, sc-58596) for other figures, anti-N-Cadherin (Proteintech, 22018–1-AP), anti-Vimentin (Proteintech, 10366–1-AP), anti-β-catenin (CST, 8480), anti-Cyclin D1 (Abcam, AB16663), anti-CD44 (Proteintech, 15675–1-AP), anti-C-Myc (CST, 5605), anti-SNAIL1(Proteintech, 13099–1-AP), anti-SNAIL2 (Proteintech, 12129–1-AP), anti-Vinculin (Proteintech, 66305–1-Ig) and anti-GAPDH (Proteintech, 10494–1-AP). Quantitative analysis of Western Blot was performed by ImageJ software. Fold changes under individual blots represented the ratio to relevant control (numbers in italic font).

Qin C., Li T., Wang Y., Zhao B., Li Z., Li T., Yang X., Zhao Y, & Wang W. (2022). CHRNB2 represses pancreatic cancer migration and invasion via inhibiting β-catenin pathway. Cancer Cell International, 22, 340.

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Western Blot Analysis of Stem Cell Markers

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The antibody Anti-BPTF for Western blot was purchased from Abcam (cat. ab72036, USA). The antibody Anti-TERT was purchased from Millipore (cat. # ABE2075, USA). The antibodies Anti-c-Kit (cat. #3308), Anti-EpCAM (cat. #2929), Anti-Cleaved Caspase-7 (cat. #8438), Anti-Cleaved Caspase-9 (cat. #7237) and Anti-Cleaved PARP (cat. #5625) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies Anti-GAPDH (cat. 10494–1-AP), Anti-β-actin (cat. 20536–1-AP), Anti-Bcl-2 (cat. 12789–1-AP), Anti-CD44 (cat.15675–1-AP) and Anti-CD133 (cat. 18470–1-AP) were purchased from Proteintech (Wuhan, China) The antibodies Anti-H3K4me3 (cat. abs136455) and Anti-H3K27me3 (cat. abs136461) were purchased from Absin Bioscience (China). The antibodies Anti-BPTF (cat. sc98404) and Anti-TERT (cat. sc7212) for IHC experiments were purchased from Santa Cruz (USA). The protein bands were detected by enhanced chemiluminescence according to the manufacturer's instructions.

Zhao X., Zheng F., Li Y., Hao J., Tang Z., Tian C., Yang Q., Zhu T., Diao C., Zhang C., Chen M., Hu S., Guo P., Zhang L., Liao Y., Yu W., Chen M., Zou L., Guo W, & Deng W. (2018). BPTF promotes hepatocellular carcinoma growth by modulating hTERT signaling and cancer stem cell traits. Redox Biology, 20, 427-441.

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Immunostaining Characterization of Neuroblastoma Cell Lines

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100,000 cells for SK-N-SH, SK-N-SHm and IC-pPDXC-63 cell lines were plated in a four-well Lab-Tek chamber (Cat# 177399PK, Thermo Fisher) 48 h before immunostaining. Cells were fixed with 4% PFA buffer, permeabilized with a 0.2% triton solution and blocked in a 1% BSA 0.1% triton solution and incubated with anti-PHOX2B (Santa Cruz Biotechnology Cat# sc-376997, RRID:AB_2813765) and anti-CD44 (Cat# 15675-1-AP, Proteintech) at 1:100. Secondary antibodies were Cy5-Anti-Mouse (Cat# 715-175-151, Jackson ImmunoResearch Labs/1:100, RRID:AB_2340820) and Cy3-Anti-Rabbit (Cat# 711-165-152, Jackson ImmunoResearch Labs/1:100, RRID:AB_2307443) and DAPI (Cat# 62248, Thermo Fisher) was diluted at 1:1000 in ProLong™ Gold (Cat# P36930, Thermo Fisher).

Thirant C., Peltier A., Durand S., Kramdi A., Louis-Brennetot C., Pierre-Eugène C., Gautier M., Costa A., Grelier A., Zaïdi S., Gruel N., Jimenez I., Lapouble E., Pierron G., Sitbon D., Brisse H.J., Gauthier A., Fréneaux P., Grossetête S., Baudrin L.G., Raynal V., Baulande S., Bellini A., Bhalshankar J., Carcaboso A.M., Geoerger B., Rohrer H., Surdez D., Boeva V., Schleiermacher G., Delattre O, & Janoueix-Lerosey I. (2023). Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma. Nature Communications, 14, 2575.

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Immunofluorescence Staining of Penile and MPG Tissues

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Slices of penile and MPG tissues were processed for immunofluorescence staining. Briefly, after the fixation with 2% formaldehyde and permeabilization with 0.5% TritonX-100, slices were incubated with primary antibodies (anti-α-SMA, anti-nNOs, anti-CD31, anti-Sca-1, and anti-CD44, ProteinTech Group, Inc) at 4 °C overnight and then incubated with CoraLite 488/594-conjugated Goat Anti-Rabbit IgG (ProteinTech Group, Inc) respectively at room temperature for 2 h in dark. Tissues were visualized under a confocal microscope (Olympus GmbH, Hamburg, Germany).

Fu Q., Song L., Li J., Yi B., Huang Y., Zhang Z., Xin Z, & Zhu J. (2023). Biodegradable nano black phosphorus based SDF1-α delivery system ameliorates Erectile Dysfunction in a cavernous nerve injury rat model by recruiting endogenous stem/progenitor cells. Journal of Nanobiotechnology, 21, 487.

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Protein Expression Analysis in Stem Cells

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Cells were washed with PBS and lysed in RIPA buffer with Protease Inhibitor Cocktail (Bimake) on ice. Cell lysates were centrifuged (12,000 rpm) at 4 °C for 20 min and then quantified using the BCA Protein Assay Kit (Beyotime). The lysate was denatured with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample loading buffer (Beyotime), followed by SDS–PAGE and electrotransfer to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). The membranes were incubated overnight at 4 °C with anti-Sestrin2 (Proteintech Group 10795-1-AP 1:1000), anti-CD44 (Proteintech Group 15675-1-1AP 1:1000), anti-Sox2 (Proteintech Group 11064-1-AP 1:1000), anti-Oct4 (Proteintech Group 11263-1-AP 1:1000), anti-CXC chemokine receptor 4 (Cxcr4) (Proteintech Group 11073-2-AP 1:1000), anti-β-catenin (Proteintech Group 51067-2-AP 1:1000) and anti-β-actin (Proteintech Group 20536-1-AP 1:1000) at the appropriate dilution and then incubated with the secondary antibody at room temperature for 2 h. The bands were visualized using BeyoECL Plus (Beyotime).

Wei J., Zheng X., Li W., Li X, & Fu Z. (2022). Sestrin2 reduces cancer stemness via Wnt/β-catenin signaling in colorectal cancer. Cancer Cell International, 22, 75.

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Osteosarcoma Tissue Microarray Immunohistochemistry

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Osteosarcoma tissue microarrays adopted in this study were purchased from Bioaitech (L714901, Xi’an, China), included 70 osteosarcoma and 1 normal tissue. The tissue microarrays were rehydrated and incubated with primary anti-CD44(Proteintech, USA 1:800) antibody, then treated with secondary antibody. Finally, the slides were colored with the DAB Kit (ZSGB-BIO, China) and photographed using the microscope.

Ji H., Kong L., Wang Y., Hou Z., Kong W., Qi J, & Jin Y. (2023). CD44 expression is correlated with osteosarcoma cell progression and immune infiltration and affects the Wnt/β-catenin signaling pathway. Journal of Bone Oncology, 41, 100487.

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Immunofluorescent Analysis of 3D Spheroid Cultures

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Spheroids were harvested at day 5 of spheroid culture, fixed with acetone, and embedded in 1% agarose. 1% Agarose blocks were dehydrated and embedded into a paraffin block. Paraffin-embedded spheroid sections (4 μm thick) were rehydrated, permeabilized with 0.1% triton X-100, blocked with 5% bovine serum albumin (BSA), and incubated with the primary antibodies: anti-GFP (Santa-Cruz Biotechnology), anti-panRAS (Millipore), anti-β-catenin (BD Bioscience), anti-CD44 (ProteinTech), anti-CD133 (ABBIOTEC), and anti-CD166 (ABBIOTEC). For secondary antibodies, Alexa Fluor 488 (Life Technologies) or Alex Fluor 555 (Life Technologies) was used. Counterstaining was done with 4′6’-diamidino 2-phenylindole (DAPI; Sigma). Gel/Mount media (Biomeda Corporation) was used for mounting. Immunofluorescent images were captured using confocal microscopy (LSM 700, Carl Zeiss). At least five fields per section were analyzed.

Ro E.J., Cho Y.H., Jeong W.J., Park J.C., Min D.S, & Choi K.Y. (2019). WDR76 degrades RAS and suppresses cancer stem cell activation in colorectal cancer. Cell Communication and Signaling : CCS, 17, 88.

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