Total protein was extracted from GFP positive flow sorted Nalm6 cells with RIPA lysis buffer and blotted with anti-FLAG (Sigma F3165; 10 µg/ml) and anti-Beta Actin (Abcam ab8227; 1:1000 dilution). Anti-mouse IgG superclonal-HRP (Invitrogen A28177; 1:5000 dilution) and anti-rabbit-HRP (Abcam ab6721-1; 1:3000 dilution) were used as secondary antibodies for FLAG and Beta Actin antibodies, respectively. Uncropped and unprocessed scans can be found in Supplementary Fig. 11 .
Ab6721 1
Manufactured by Abcam
Sourced in United Kingdom
Ab6721-1 is a primary antibody used in various research applications. It is a mouse monoclonal antibody that recognizes a specific target antigen. The core function of this product is to serve as a tool for researchers to detect and study the target antigen in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab6046, by Abcam (1 mentions)
Ab41651, by Abcam (1 mentions)
Ab97046, by Abcam (1 mentions)
Protecease 50, by G Biosciences (1 mentions)
Ab6808, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
P akt, by Abcam (1 mentions)
Ab8932, by Abcam (1 mentions)
Ab64148, by Abcam (1 mentions)
Ab131438, by Abcam (1 mentions)
P erk, by Abcam (1 mentions)
Ab32537, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Sc 6836, by Santa Cruz Biotechnology (1 mentions)
Ab6741, by Abcam (1 mentions)
6 protocols using ab6721 1
1
GFP-sorted Nalm6 Protein Extraction
2
Immunoblotting Analysis of STAT1 Signaling
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Cell pellet was lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) with 1X proteCEASE-50 (#427P;G-Biosciences, St.Louis, MO,USA) 1μM PMSF. Protein was quantified by Bicinchoninic acid (BCA) assay. The protein was resolved on SDS-PAGE and transferred on PVDF membrane. Membrane was kept for blocking in 5% skimmed milk in 1X TBS-Tween20. Thereafter, the membrane was incubated in primary antibody (1:1000) overnight. HRP-conjugated secondary antibody incubation was used for 1 hour and then membrane was washed thrice with 1X TBST and developed by using Super-Signal developing reagent as HRP substrate. The anti-STAT1 (#9172 Cell Signaling technology), anti-phospho-Y701-STAT1 (#9167 Cell Signaling technology), anti-SOCS-5 (#sc-5607, Santa cruz biotechnology) anti-JEV NS1 (#ab41651; Abcam) and anti-β-tubulin (#ab6046; Abcam) primary antibodies were used after diluting in 5% BSA in TBST buffer. HRP-conjugated anti-rabbit (#ab6721-1; Abcam) and anti-mouse (#ab97046, Abcam) secondary antibody was used in 1:50,000 dilutions.
3
Analyzing FANCD2 Foci and FANCA Expression
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Analyses of FANCD2 foci were performed by immunofluorescence of primary fibroblasts or iPSCs treated for 16 h with 200 nM of MMC. After MMC treatment, cells were stained with rabbit polyclonal anti-FANCD2 (Abcam, ab2187-50) as previously described (Hotta & Ellis, 2008 (link); Raya et al, 2009 (link)). Cells with more than ten foci were scored as positive. FANCA expression was analyzed by Western blot (Raya et al, 2009 (link)) using the following antibodies: hFANCA (ab5063 Abcam) and anti-beta Actin to mouse antibody (ab6276, Abcam) as control. Goat polyclonal antibody to rabbit IgG (HRP; ab6721-1; Abcam) and sheep polyclonal antibody to mouse IgG—H&L (HRP; ab 6808, Abcam) were used as secondary antibodies. Protein quantification was done with Image J software.
4
Western Blot Analysis of Signaling Proteins
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After 48 hours of transfection, the cells in six-well plates were collected and placed on ice. To extract the proteins, RIPA lysate with protease inhibitor was used. BCA method was used to determine the protein concentration. Then we added about 20 μg protein to each well of a vertical electrophoresis tank after being heated at 95 °C for 5 min. Following that, the protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. The primary antibodies used in the current study were list as follows: ARHGEF39 (1:1000, cat.no. ab67211, Abcam), AKT (1:500, cat. no. ab64148, Abcam), p-AKT (1:500, cat.no. ab8932, Abcam), ERK (1:1000, cat.no. ab32537, Abcam), p-ERK (1:1000, cat.no. ab131438, Abcam). Following that, the membrane was rinsed with TBST 35 min and incubated with suitable secondary antibodies at ambient temperature. Then the protein bands were washed and developed with enhanced chemiluminescence western blot detection kit. The gray value was scanned by the QUANTITY ONE software and the relative expression of each protein was calculated with GAPDH as the internal reference.
5
Western Blot Analysis of TIMP-3 Protein
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The GCs protein extract (30 μg) was separated by SDS-PAGE using 13% gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA). The membrane was blocked overnight at 4°C with 5% skimmed milk and then washed for 10 min with the wash buffer (0.1% v/v Tween 20, 50 mM Tris-HCl and 200 mM NaCl; pH 7.6). The transferred membrane was incubated for 2 h with first antibodies (diluted 1:1,000 in blocking buffer) recognizing the active forms of TIMP-3 (sc-6836, Santa Cruz Biotechnology Inc., Dallas, TX) and β-actin (sc-47778, Santa Cruz Biotechnology Inc., Dallas, TX). After binding the antibodies, the membranes were washed three times for 15 min each with TBS-T (40 mM Tris-HCl pH 7.4, 25 mM NaCl, 0.1 % Tween-20) buffer and incubated for 2 h with horseradish peroxidase (HRP)-conjugated anti-goat (ab6721-1, Abcam, Cambridge, UK) or anti-mouse (ab6741, Abcam, Cambridge, UK) secondary antibodies (Abcam, Cambridge, UK; diluted 1:5,000 in blocking buffer). The transferred membranes were reacted with the enhanced chemiluminescence (ECL) detection reagent in the dark and then exposed to X-ray film for about 10 min; protein expression was normalized to that of β-actin protein, which acts as an internal control target, using the Alpha Innotech software (San Leandro, CA).
6
Immunohistochemical Analysis of Glioma Tissues
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After antigen retrieval, the sections of glioma tissues were incubated overnight at 4°C with rabbit primary antibodies including anti-Ki-67 (ab18850, 1:200, Abcam), anti-PD-L1 (#13,684, 1:200, CST), and anti-p-ERK (#4370, 1: 200, CST). After washing, the sections were incubated for 30 min at room temperature with secondary IgG antibody (#ab67211, 1:400, Abcam), followed by visualization using DAB and hematoxylin. Stained sections were then observed with an optical microscope, and images were photographed for analysis. For image analysis, the ‘IHC Profiler’ plugin of the ImageJ software was utilized for deconvolution [21 (link)].
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