For SDS‐PAGE/Western blot analysis, the protein lysates were prepared by lysis with Tris‐HCl buffer, NP‐40 (1%), PMSF (1 mmol/L), and inhibitor cocktail (Roche, Switzerland). Protein samples were separated by 4–20% SDS‐PAGE (BioRad, Berkeley, CA) and transferred onto PVDF membrane. Protein detection was performed with polyclonal antibodies: anti‐DNA PKC (1:200, abcam), anti‐ChK2 (1:1000, Cell Signaling), anti‐phospho ChK2 (1:1000, Cell Signaling), anti‐ATM (1:1000, abcam, Cambridge, UK), anti‐phospho ATM (1:1000, abcam), anti‐TS (1:200, abcam), anti‐ MITF (1:1000, abcam), anti‐TK1 (1:10000, abcam), anti‐Caspase3 (1:1000, Cell Signaling), anti‐cleaved Caspase3 (1:1000, Cell Signaling), anti‐PARP (1:1000, Cell Signaling), and anti‐cleaved PARP (1:1000, Cell Signaling). The secondary IgG coupled to HRP (1:2000, Cell Signaling) was visualized with enhanced chemiluminescence (ECL+, GE Healthcare, UK). Equal protein loading was controlled using GAPDH‐specific antibody (Ambion, Life Technologies, Carlsbad, CA) and secondary goat antimouse IgG linked to HRP (abcam). The experiments were carried out three times in triplicates. The bands were detected using the ImageQuant LAS 4010 camera system (GE Healthcare).
Anti mitf
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-MITF is a primary antibody that recognizes the MITF (Microphthalmia-associated transcription factor) protein. MITF is a key transcription factor involved in the development and function of melanocytes, the pigment-producing cells in the skin. This antibody can be used to detect and study the expression of MITF in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Anti chk2, by Cell Signaling Technology (1 mentions)
Anti phospho chk2, by Cell Signaling Technology (1 mentions)
Anti phospho atm, by Abcam (1 mentions)
Anti tk1, by Abcam (1 mentions)
Gapdh specific antibody, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4010 camera system, by GE Healthcare (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Odyssey clx system, by LI COR (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Fluorescein conjugated secondary antibody, by LI COR (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab12039, by Abcam (1 mentions)
Anti phospho mek1 2, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
12 protocols using anti mitf
1
SDS-PAGE and Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of MITF in Murine RPE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPE sheets isolated from two-month-old C57BL/6 mice were lysed in a solution containing RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, China) and protease inhibitors (Beyotime Institute of Biotechnology). After sonication, protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Life Science, Hercules, CA, USA) as described previously.31 The membranes were blocked for two hours in a blocking buffer containing 5% non-fat milk in PBS at room temperature and incubated overnight with primary antibodies at 4°C. Primary antibodies were anti-MITF (1:1000, ab12039; Abcam, Cambridge, MA, USA) and anti-GAPDH (KC-5GC, 1:5000; Aksomics, Shanghai, China). After washing with Tris-buffered saline solution containing Tween 20, the membranes were incubated with fluorescein-conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) at room temperature for two hours and analyzed by the Odyssey CLx System (LI-COR Biosciences).
3
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from cells using PhosphoSafe Extraction Reagent (Novagen). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.2-μm PVDF membranes. The membranes were blocked in 5% BSA in TBS-Tween 20 (TBS-T) for 2 h and incubated overnight at 4°C with the following primary antibodies: anti-ADCY1 (Santa Cruz), anti-phospho-MEK1/2 (Cell Signaling Technology), anti-total-MEK1/2 (Cell Signaling Technology), anti-phospho-ERK1/2 (Cell Signaling Technology), anti-total-ERK1/2 (Cell Signaling Technology), anti-phospho-CREB (Abcam), anti-total-CREB (Proteintech), anti-MITF (Abcam), anti-N-cadherin (Proteintech), anti-E-cadherin (Proteintech), anti-vimentin (Proteintech), and anti-slug (Proteintech). After washing with TBS-T for 3 times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (MBL) for 1 h. Bands were visualized with ECL SelectTM Western Blotting Detection Reagent (GE Healthcare), and bands intensities were quantified using ImageJ software.
4
Antibody Panel for Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in this study, including anti-actin, anti-PKA, anti-GSK3β, anti-p-GSK3β, anti-mouse, and anti-rabbit secondary antibody were purchased from GeneTex (Irvine, CA, USA). Antibodies including anti-p-ERK and anti-CREB were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies including anti-p-CREB, anti-MITF, and anti-p-MITF were purchased from Abcam (Cambridge, UK). Other primary and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
5
Quantifying MITF, CRALBP, and RDH5 in ARPE19 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from Mitf or Control siRNA-treated ARPE19 cells, and EGFP or Mitf-lentivirus-infected ARPE19 cells were subjected to Western blotting with anti-MITF (Abcam, 12039), anti-CRALBP (Thermo scientific, MA1-813), anti-RDH5 (Santa Cruz, sc-98348), anti-TUBULIN (Santa Cruz, sc-5286) antibodies. Western blots were prepared and performed according to a published procedure20 (link).
6
Immunostaining of Embryonic Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryo heads (E10.5–E16.5) were freshly dissected, cryopreserved in OCT compound, and sectioned along the coronal and sagittal plane, followed by 4% formaldehyde fixation solution at 4°C for 1 h. A total of 16–18 μm embryonic tissue sections were then processed for immunostaining by phosphate buffered saline (PBS) rinse, blocked for 1 h in 5% BSA plus 0.1% triton at room temperature (RT), incubated with primary antibodies overnight at 4°C, followed by 1 h incubation at RT with fluorescence-conjugated secondary antibodies (Jackson Laboratory). The slides were mounted using DAPI mounting medium (Vector). The primary antibodies used in the experiments were anti-PH3 (1:500, Millipore), anti-Ki67 (1:400, Millipore), anti-MiTF (1:400, Abcam), anti-AP2β (1:300, Cell Signaling Technology), anti-CD31 (1:500, BD), anti-PDGFRβ (1:500, Abcam), anti-Sox9 (1:300, Millipore), anti-FoxD3 (1:300, Cell Signaling Technology), and anti-Sox10 (1:300, Cell Signaling Technology). Hyaloid vessels and retinal vessels were stained using fluorescein isolectin B4 (1:500, Vector Laboratories) at 4°C overnight. DAPI (Sigma and Vector Laboratories) was used for nuclear staining.
7
Modulation of Mitf Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hNrg1–1 (25 ng/mL) (Cell Signaling Technology, Catalog # 5218SC); VPS34-IN1 (1 μM) (EMD Millipore, Catalog # 5326280001); YM201636 (2 μM) (Cayman Chemical, Catalog # 13576–1); Canertinib (5 μM) (Selleck Chemicals LLC, Catalog #S1019); MK-2206 (1 μM) (Selleck Chemicals LLC, Catalog #S1078); Torin (5 μM) (APExBIO, Catalog # A8312); Enzastaurin (5 μM) (Selleck Chemicals LLC, Catalog #S1055); Trametinib (5 μM) (Selleck Chemicals LLC, Catalog #S2673); KT5720 (Sigma-Aldrich, Catalog # 420323); Sch772984 (5 μM) (APExBIO, Catalog #B5866); BIO (10 μM) (GSK3, Sigma-Aldrich Inc Cat# B1686); Rapamycin (0.27 μM) (mTorc1, Thermo Scientific Chemicals, Catalog #J67452.XF). Rabbit polyclonal Mitf antibody was raised in house (Animal # 7414, # 7416) against antigens DLVNRIIKQEPVLENCSQE (N-term) and CGTMPESSPAYSIPRKMGSNLEDILMD (C-term), respectively, each independently conjugated to KLH (Thermo Scientific, Cat# 77671). Commercial antibodies: anti-Paxillin (BD Biosciences #610051). Anti-Mitf (Abcam, ab122982), anti-GAPDH (Fitzgerald Cat # 10R-G109a), anti-GFAP (Aviva Cat # OAPC00115), anti-GFAP (Aviva Cat# OAEB01041), anti-S100 (Abcam Cat# ab868), anti-Sox10 (R and D systems, Cat# AF2864). Alexa-conjugated secondary antibodies (Thermo Fisher Scientific).
8
Characterization of Melanoma Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used: anti-MITF (Abcam) along with APC-conjugated goat anti-rabbit (Santa Cruz Biotechnology) and anti-Melan-A (DAKO) along with FITC-conjugated goat anti-mouse (BD Pharmingen). Typically, 30,000 cells/sample were analyzed. Appropriate isotype controls were used. To exclude dead cells from the analysis, LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (Invitrogen) was used. Acquisition was performed using FACSVerse flow cytometer (Becton Dickinson) and analyzed using BD Cell Quest software.
9
Immunohistochemical Characterization of Retinal Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ROs were fixed in 4% paraformaldehyde for 30 min and imbedded in O.C.T. compound and sectioned into 10 μm slices. The cryosections were blocked with 0.5% Triton X-100 in 4% bovine serum albumin (BSA) for 1 hr. After that, sections were incubated in primary antibodies (diluted in 4% BSA supplied with 0.5% Triton X-100) at 4°C overnight. The following primary antibodies were used: anti-OTX2 (1:200, Cat.# ab183951; Abcam), anti-PAX6 (1:200, Cat.# 901301; Biolegend), anti-SOX2 (1:200, Cat.# sc-365823; Santa Cruz), anti-HuC/D (1:100, Cat.# A21271; Invitrogen), anti-MITF (1:100, Cat.# ab3201; Abcam), anti-GFAP (1:200, Cat.# sc-33673; Santa Cruz), anti-Sox9 (1:200, Cat.# 711048; Invitrogen), anti-RxRγ (1:100, Cat.# sc-365252; Santa Cruz), and anti-Ki67 (1:200, Cat.# ab15580; Abcam). After washed with phosphate-buffered saline, cryosections were stained with Alexa Fluor-conjugated secondary antibodies (diluted 1:500, Invitrogen) for 1 hr at room temperature in the dark.
10
Western Blot Analysis of Melanogenesis Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with a RIPA cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, a protease inhibitor cocktail, 50 mM NaF and 0.2 M NA3VO4). Proteins were separated by a 10% sodium dodecyl sulfate-polyacrylamide gel, and transferred onto a nitrocellulose membrane (Whatman International Ltd., Kent, UK). After blockage with Tris-buffered saline containing 0.1% Tween 20 and 5% skim milk, the membranes were incubated with anti-MITF (Abcam, Cambridge, MA, USA), anti-TYR (Abcam), anti-Rap1a (Novus, Centennial, CO, USA), anti-Akt (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt (Cell Signaling Technology), anti-GSK3β (Cell Signaling Technology), anti-phospho-GSK3β (Cell Signaling Technology) and anti-actin (Sigma-Aldrich, Burlington, MA, USA) antibodies. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. The target protein’s signals were developed with the enhanced chemiluminescence kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA).
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!