- Rafik Karaman added an answer:1What is the most efficient method to label lactoferrin with FITC?
Recently, I want to label protein lactoferrin with FITC and find how lactoferrin interacts with the bacteria, but I did not find an efficient method.
The following paper covers the answer to your question:
Infect Immun. 2001 May; 69(5): 3372–3381.
Characterization of Binding of Human Lactoferrin to Pneumococcal Surface Protein A
Anders Håkansson,1,2,* Hazeline Roche,1 Shaper Mirza,1 Larry S. McDaniel,3 Alexis Brooks-Walter,1,† and David E. Briles1
Editor: E. I. Tuomanen
MATERIALS AND METHODS
Protein markers were from Amersham Pharmacia Biotech (Piscataway, N.J.). NBT (nitroblue tetrazolium) and BCIP (5-bromo-4-chloro-3-indolylphosphate) were from Fisher Scientific (Atlanta, Ga.). Alkaline phosphatase (AP)-conjugated streptavidin, biotin-conjugated goat anti-mouse immunoglobulin (Ig), and biotin-conjugated goat anti-rabbit Ig antibodies were from Southern Biotechnology Associates (Birmingham, Ala.). Fluorescein isothiocyanate (FITC)-conjugated streptavidin, R-phycoerythrin (RPE)-conjugated streptavidin, FITC-conjugated rabbit anti-mouse Ig antibodies, and FITC-conjugated goat anti-rabbit Ig antibodies were from Dako A/S (Rothskild, Denmark). Bacto-Todd Hewitt medium and yeast extract were from Difco Laboratories (Detroit, Mich.). Human and bovine milk lactoferrin and human transferrin were from Sigma Chemical Co (St. Louis, Mo.).
Recombinant human lactoferrin was kindly provided by Arne Forsgren, Department of Medical Microbiology, Lund University, University Hospital Malmö, Malmö, Sweden.
Monoclonal anti-PspA antibodies Xi126 and XiR278 have been previously described (38). Polyclonal anti-PspC serum was raised by immunization with a truncated PspC molecule from S. pneumoniae L81905expressed in Escherichia coli (19). Polyclonal anti-PspA family 1 antiserum was pooled from two rabbits immunized with recombinant PspA/L82016 (clade 1) or recombinant PspA/Rx1 (clade 2) expressed in E. coli. Anti-PspA family 2 antiserum was produced by pooling serum from two rabbits immunized with either recombinant PspA/V-024 (clade 3) or recombinant PspA/V-032 (clade 4) expressed in E. coli. To this pool, recombinant PspA/Rx1 was added to reduce its cross-reactivity with family 1 (clade 1 and 2) PspAs. Approximately 10 μg of the purified PspA proteins was injected subcutaneously into a rabbit twice on consecutive weeks, and blood was collected 10 days after the last injection. The primary immunization was given with Freund's complete adjuvant, and the booster immunization was given in saline.
The strains and plasmids used in this study are described in Table Table1.1. The pneumococcal strains were stored at −80°C in fetal calf serum, transferred to blood agar plates, and incubated at 37°C in a 5% CO2atmosphere overnight. Colonies grown on blood agar were used to inoculate liquid growth medium (Todd-Hewitt medium containing 0.5% yeast extract [THY]). Upon reaching late log phase, the bacteria were harvested by centrifugation at 1,500 × g for 15 min and suspended in 60 mM phosphate-buffered saline (PBS, pH 7.2). The bacterial concentration was estimated by interference contrast microscopy (TE Leitz Ortolux II microscope with interference contrast equipment; Leitz, Wetzlar, Germany) using a Bürker chamber and confirmed by counting viable cells. Appropriate dilutions of the bacteria were suspended in PBS.
The pspC gene was insertionally inactivated in S. pneumoniae D39. An internal fragment of pspC was amplified using PCR and cloned into pSF143 (51). The ligated plasmid was electroporated into E. coliDH5α, and clones were selected for tetracycline resistance. The plasmid, which contained the internal fragment of pspC, was isolated from recombinant E. coli using standard procedures and transformed into S. pneumoniae D39 (30). Tetracycline-resistant recombinants were screened by both Southern hybridization and Western blotting to confirm inactivation of pspC. Lysate from the strain containing the insertionally inactivated pspC (TRE118) was transformed into JY53 (erythromycin resistant, pspA negative) to create a mutant that lacked both pspA and pspC (TRE121) (23, 59).
Binding of lactoferrin and transferrin to bacterial cells.
Purified lactoferrin from human or bovine milk, recombinant human lactoferrin, and human transferrin were biotinylated using the Roche biotin labeling kit according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, Ind.).
Bacteria were grown in THY medium (S. pneumoniae) or on chocolate agar plates (Moraxella catarrhalis) and suspended in PBS at a concentration of approximately 5 × 108 bacteria/ml. The bacterial suspension (100 μl) was mixed with 0.5 to 10 μl of biotinylated protein (2-mg/ml stock solution in PBS) for 30 min at room temperature and washed by centrifugation at 1,500 × g for 5 min in PBS. FITC-conjugated streptavidin (1:100 dilution in PBS) was added for an additional 30 min at room temperature, and after a final wash in PBS, the cells were inspected by epifluorescence and laser scanning confocal microscopy using MRC-1024 confocal equipment (Bio-Rad Laboratories, Hemel-Hampstead, United Kingdom) attached to a Nikon Eclipse E800 upright microscope (Nikon, Tokyo, Japan). The binding was quantitated by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson Biosciences, Rutherford, N.J.).
Hoping this will be helpful,
- Agnieszka Barbara Najda added an answer:4What is the retention time of lactoferrin when using TFA and acetonitrile as mobile phase in a reverse phase C18 column?
when I use C18 Reverse phase column , TFA and acetonitrile as mobile phase , gradient elution at a flow rate of 0.5 ml/min , the retention time is around 3.58 mins and I am not sure about the peaks that are eluted , when i compare it with the whey I get the same pattern of peaks but at aroun 5.45 mins. Can someone help me how to analyse with the same. Thank you
I agree with the opinion Jandirk Sendker.
But also important are the parameters for whom he recalls Wołodymyr Skalka.
Are internal pattern, also you get kakie results? The matrix of whey may also cause some displacement. But what is most important, then check whether your gradjent is well configured.Following
- Mohammed Al Marjani added an answer:6What is the effect of lactoferrin on MDR bacteria?
What is the effect of lactoferrin on Multidrug resistant bacteria?
Thank you Traci for your helpingFollowing
- Aletheia Lima Souza added an answer:4Does anyone know if I can reverse denaturation by urea (7M)?
I am working with lactoferrin and over my protocol for expression in vitro I have been used urea (7M) buffer for denaturation, however, I would like to proceed an assay with my lactoferrin and it should be intact. Can I reverse denaturation by urea of my lactoferrin? I appreciate any idea.
Hey all, I performed my assay, and right now I am waiting for the results.
Thank you guys for your reply. It was ver helpful.
Obrigada Noelita, eu estou plotando os dados pra ver se a proteina mantem a atividade. Muito bom ter noticias suas. Bjao amiga :-).
Jorge, amigo, valeu a dica. QQ coisa bato um fio pra ti. A gnt segue aqui com os experimentos. BjaoFollowing
- Vesna Karapetkovska Hristova added an answer:6What are the conditions of RP-HPLC for lactoferrin and lysozyme detection?
Can anyone suggest how I can prepare samples (cow's milk) and protein standards (lactoferrin and lysozyme) for RP-HPLC? What conditions (mobile phase, gradient, operating temperature, flow rate) I can use for RP-HPLC whey protein detection?
I m wishing to you a BIG success in this hard work, but believe me, here in Macedonia also we have problems with lactoferin detection in colostrum, in normal milk etc, and also a lot of money for this....I agree with d-r Bianchi, try to do this by ELISA instrument, ask in your local laboratories........best
- Prof. Ravi K. Sharma added an answer:5Does Bovine lactoferrin have antioxidant effect?
lactoferrin is used as immunostimulant
A Google search on "Bovine lactoferrin antioxidant pdf returned about 2,14,000 hits
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- Maged El-Ashker added an answer:2Does any body know of a Company that produces bovine lactoferrine, protected fish oil and equine recombinant IL-7?
Shall any body inform me by the source for these products, I appreciate his/her kind help.
Thank you so much Mr. Saad for your Answer, I will try this.Following
Lactoferrin (LF), also known as lactotransferrin (LTF), is a multifunctional protein of the transferrin family. Lactoferrin is a globular glycoprotein with a molecular mass of about 80 kDa that is widely represented in various secretory fluids, such as milk, saliva, tears, and nasal secretions.