Lm cell envelope proteins were extracted as described in Section 3.5 . Twenty μg of protein were loaded and subjected to 12% SDS-PAGE. Western blots were probed with HCl- and NaOH-LMGs vaccinated or PBS-injected antisera (6 weeks, 1:1000) in TBS-T buffer containing 5% non-fat dry milk as primary antibodies. Goat anti-mouse IgG conjugated alkaline phosphatase (1:7000; Millipore Sigma) was used as a secondary antibody. Detection was developed using NBT Substrate Powder containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Thermo Fisher Scientific, Waltham, MA, USA).
5 bromo 4 chloro 3 indolylphosphate
Manufactured by Thermo Fisher Scientific
Sourced in United States
5-bromo-4-chloro-3-indolylphosphate is a chromogenic substrate used in biochemical applications. It is a chemical compound that undergoes a color change reaction when acted upon by certain enzymes, allowing for detection and visualization of those enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 1260 infinity, by Agilent Technologies (1 mentions)
Coupling reagents, by GL Biochem (1 mentions)
Ni sepharose 6 fast flow beads, by GE Healthcare (1 mentions)
Dipeptides, by Merck Group (1 mentions)
Amino acids, by GL Biochem (1 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (1 mentions)
Smz745t microscope, by Nikon (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Ultraflextreme maldi tof tof mass spectrometer, by Bruker (1 mentions)
Microflex maldi tof mass spectrometer, by Bruker (1 mentions)
Skimmed milk, by HiMedia (1 mentions)
Nitro blue tetrazolium chloride, by Thermo Fisher Scientific (1 mentions)
Anti ferroportin, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti dmt1, by Santa Cruz Biotechnology (1 mentions)
Anti hepcidin, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Anti transferrin, by Santa Cruz Biotechnology (1 mentions)
Amersham image scanner, by GE Healthcare (1 mentions)
5 protocols using 5 bromo 4 chloro 3 indolylphosphate
1
Western Blot Analysis of Lm Envelope Proteins
2
Peptide-based Protein Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
General chemicals and reagents were purchased from Fisher Acros (Hampton, NH, USA). Isothiocyanobenzyl-NTA was purchased from Biomass (Rockville, MD, USA). Tentagel S NH2 resin was purchased from RAPP polymer (Tuebingen, Germany). DynabeadsTM MyOneTM Streptavidin C1, Streptavidin-alkaline phosphatase (SAAP), and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) were obtained from Thermofisher (Hampton, NH, USA). Amino acids, coupling reagents, and biotin were purchased from GL-Biochem. BSA (Shanghai, China), lysozyme, and dipeptides were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ni Sepharose 6 FastFlow beads were obtained from GE Healthcare (Chicago, IL, USA).
MALDI-TOF-MS and MALDI-TOF-MS/MS analysis were performed by a Bruker Micro-flex MALDI-TOF mass spectrometer (Billerica, MA, USA) and a Bruker Ultra-flextreme MALDI-TOF/TOF mass spectrometer. The HPLC analysis and HPLC purification were performed using Agilent 1260 Infinity (Agilent, Santa Clara, CA, USA). Positive bead (blue beads) screening was performed with a Nikon SMZ 745T microscope (Nikon, Tokyo, Japan).
MALDI-TOF-MS and MALDI-TOF-MS/MS analysis were performed by a Bruker Micro-flex MALDI-TOF mass spectrometer (Billerica, MA, USA) and a Bruker Ultra-flextreme MALDI-TOF/TOF mass spectrometer. The HPLC analysis and HPLC purification were performed using Agilent 1260 Infinity (Agilent, Santa Clara, CA, USA). Positive bead (blue beads) screening was performed with a Nikon SMZ 745T microscope (Nikon, Tokyo, Japan).
3
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sample containing 60-100 μg of protein was equally loaded on two parallel gels. The proteins depending upon the molecular weight were resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membrane (PVDF) (Bio-Rad Laboratories, Inc., Hercules, CA)as described elsewhere [26] (link). To prevent non-specific binding, PVDF membrane was incubated with 5% skimmed milk (HiMedia Laboratories Pvt. Ltd., Mumbai, India) for 60 min. Incubation of membrane was done with the primary antibodies [anti-transferrin, anti-ferroportin, anti-DMT-1, anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX), anti-ceruloplasmin or anti-hepcidin (Abcam, Cambridge, UK)] for overnight followed by 1 h incubation with anti-goat/anti-rabbit/anti-mouse (Santa Cruz Biotechnology Inc., Dallas, TX) secondary antibody conjugated with alkaline phosphatase (AP). Since the secondary antibody used in the experiment was AP-conjugated, one gel was processed for target protein and other for respective loading control. The protein bands were developed using AP specific chromogenic substrates, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chloride (Thermo Fischer Scientific, Waltham, MA). Band density ratio was calculated in relation to β-actin using Alpha Imager software.
4
Immunodetection of Fusion Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pHluorin and mCystaTag–Q47P fusions were immunodetected on nitrocellulose sheets following protein separation by 12% (w/v) SDS‐PAGE in reducing conditions. pHluorin was detected with mouse anti‐GFP monoclonal antibodies (Clontech Laboratories, Mountain View, CA, diluted 1:5000) and goat anti‐mouse secondary antibodies conjugated to alkaline phosphatase (Sigma‐Aldrich, diluted 1:5000). Q47P and mCystaTag were detected with custom‐made rabbit anti‐SlCYS8 polyclonal antibodies (Robert et al., 2013 ; Agrisera, Vännäs, Sweden, diluted 1:5000) and goat anti‐rabbit secondary antibodies conjugated to alkaline phosphatase (Sigma‐Aldrich, diluted 1:5000). Nonspecific binding on nitrocellulose sheets was prevented by incubation in blocking solution (5% w/v skim milk powder in PBS, containing 0.025% v/v Tween‐20), which also served as antibody dilution buffer. Protein signals were developed with the alkaline phosphatase substrate 5‐bromo‐4‐chloro‐3‐indolyl phosphate and nitro blue tetrazolium as a colour indicator (Life Technologies, Burlington ON, Canada). Densitometric analysis of the fusion proteins was performed using the Phoretix 2D Expression software, v. 2005 (Nonlinear USA, Durham, NC) on non‐saturated immunoblot images digitalized with an Amersham Image Scanner (GE Healthcare). All measurements were made with leaf extracts from three independent (plant) replicates.
5
Immunodetection of Plant Stress Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T6R, P2V and ß-glucanases (PR-2 proteins) were immunodetected in total soluble protein extracts prepared from the fifth leaf of 30 cm-tall plants, down from the apex. Leaf soluble proteins were extracted in mild conditions as described earlier49 (link) and resolved by 12% (w/v) SDS-PAGE using the Bio-Rad Mini Protean III Electrophoresis Unit™ (Bio-Rad). The proteins were electrotransferred onto Hybond ECL nitrocellulose sheets (GE Healthcare) using the Bio-Rad Mini-transfer Unit™, according to the supplier’s instructions. T6R and P2V were immunodetected with commissioned polyclonal IgG raised in rabbits against SlCYS8 (AgriSera), ß-glucanases with commercial polyclonal IgG raised in rabbits against tobacco PR-2 proteins (AgriSera), and primary IgG with goat anti-rabbit IgG conjugated to alkaline phosphatase. Protein–antibody complexes were visualized using the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium for colour development (Life Technologies). Densitometric analysis of the protein signals on nitrocellulose sheets was performed with the Phoretix 2D Expression software, v. 2005 (NonLinear USA) after scanning the immunoblots with an Amersham Image Scanner digitalizer (GE Healthcare). All immunodetections involved three independent (biological) replicates to allow for statistical analyses.
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!