Share Give access Share full text access. Share full text access. Please review our Terms and Conditions of Use and check box below to share full-text version of article. Abstract In this unit, protocols describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters. Citing Literature.
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Special Considerations for Proteoglycans and Glycosaminoglycans and Their Purification
Previous Figure Next Figure. Email or Customer ID. Forgot password? Old Password. New Password. Password Changed Successfully Your password has been changed. Stop mixture was added to terminate the sequencing reactions. Samples were electrophoresed for about 4 hours at 60 volts, constant current. After electrophoresis, the gels were dried in vacuo and autoradiographed. The autoradiographs were analyzed manually. Based on the sequence data, new sequencing primers were prepared and the process was repeated until the entire gene had been sequenced.
All sequences were confirmed a minimum of three times in both orientations. Percent homology values for nucleotide sequences were calculated by the program NALIGN, using the following parameters: weight of non-matching nucleotides, 10; weight of non-matching gaps, 10; maximum number of nucleotides considered, 50; and minimum number of nucleotides considered, A value of 10 was selected for both the open gap cost and the unit gap cost. The differential display PCR amplification products described in Example 9 contained a major band of approximately base pairs.
Positive clones were subjected to nested primer analysis to obtain the primer sequences necessary for amplifying the full length cDNA. ID NOs: 11, 12, 29, 30 and One of those positive clones selected for further study was designated clone Sequence analysis of the full-length cDNA in clone , determined by nested primer analysis, showed that clone contained the original base-pair fragment identified by differential display PCR Clone base pairs; SEQ ID NO: 2 contains an open reading frame of base pairs encoding a protein having amino acids SEQ.
ID NO: 1. The termination codon, TGA, occurs at nucleotides ID NO: 1, respectively. Two potential cAMP- and cGMP-dependent protein kinase phosphorylation sites, Ser and Thr, were found at amino acid positions and , respectively. There were five potential protein kinase C phosphorylation sites, Ser or Thr, at amino acid positions 3, , , , In addition, two highly conserved putative LIM domains were found at amino acid positions and RLMP also designated has Northern blot analysis showed a 1.
Graphs may be shown with data normalized to the maximum value for each parameter to allow simultaneous graphing of nodule counts, mineralized areas and osteocalcin. Graphs may be shown normalized to either day 0 or negative controls and expressed as fold-increase above control values. Statistical significance was evaluated using a one-way analysis of variance with post-hoc multiple comparison corrections of Bonferroni as appropriate. Billingsley ed. Polyclonal antibodies were prepared according to the methods of England, et al.
Acta, , and Timmer, et al. Protein was harvested from the transfected cells according to the method of Hair, et al. USA, Culture supernatant was removed by aspiration and the flasks were overlayed with 3. RNA was purified by a slight modification of standard methods, for example, see Sambrook, et al. Briefly, the 0. RNA concentrations were derived from optical densities. ID NO: 6. ID NOs: 12, 15, 16, 17, 18, 27 and Sequences were confirmed a minimum of two times in both directions.
ID NO: 7. Based on Northern blot experiments, it was determined that LMP-1 is expressed at different levels by several different tissues, including human heart muscle. A human heart cDNA library was therefore examined. Filter membranes were overlaid onto the plates for two minutes. Double-positive plaque-purified heart library clones, identified by autoradiography, were rescued as lambda phagemids according to the manufacturers' protocols Stratagene, La Jolla, Calif.
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Restriction digests of positive clones yielded cDNA inserts of varying sizes. Inserts greater, than base-pairs in length were selected for initial screening by sequencing. Those: inserts were sequenced by standard methods as described in Example One clone, number 7, was also subjected to automated sequence analysis using primers corresponding to SEQ. ID NOS: , 16 and Overlapping regions of the MG63 human osteosarcoma cell cDNA sequence and the human heart cDNA clone 7 sequence were used to align those two sequences and derive a complete human cDNA sequence of base-pairs.
The overlapping regions of the two sequences constituted approximately base-pairs having complete homology except for a single nucleotide substitution at nucleotide in the MG63 cDNA SEQ. ID NO: 8. The resulting sequence is shown in SEQ. ID NO: 9. II NO: 9 is The open reading frame in SEQ. Second-strand synthesis is performed according to the method of Gubler, et al.
Adaptor-ligated double-stranded cDNA was then ready to be specifically cloned. The first round PCR product was approximately base-pairs in length whereas the nested PCR product was approximately base-pairs.
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The base-pair clone Example 17; SEQ. Clone 7 Example 18; SEQ. Due to the single nucleotide substitution in Clone 7 relative to the base-pair PCR sequence and the original rat sequence a stop codon at translated base-pair resulted. Because of this stop codon, a truncated short protein was encoded, hence the name LMP-1s. This was the construct used in the expression vector SEQ. The vector was recovered and the base-pair restriction fragment ent was then hgated into the linearized pHis-ATG.
The ligated product was cloned and amplified. Rat Calvarial cells were isolated and grown in secondary culture according to Example 1. Cultures were either unstimulated or stimulated with glucocorticoid GC as described in Example 1. Mineralized nodules were visualized by Von Kossa staining, as described in Example 3. The effect was less than that observed with GC stimulation, most likely due to sub-optimal doses. The ligated DNA was gel purified, extracted and used to transform E.
Positive agar colonies were picked, digested with NotI and ApaI and the restriction digests were examined by gel electrophoresis. Stock cultures were prepared of positive clones. Superfect was added 7. Afterward, the cells were gently washed once with sterile PBS and the appropriate normal incubation medium was added. Results demonstrated significant bone nodule formation in all rat cell cultures which were induced with pCMV Similarly, when cultures were transfected with pCMV reverse , no nodules were observed.
The aspirated marrow cells were washed in alpha MEM, centrifuged, and RBCs were lysed by resuspending the pellet in 0. The animals were scarified at weeks, at which time the discs, or surgical areas were excised and fixed in 0.
Experiments were also performed using devitalized guanidine extracted demineralized bone matrix Osteotech, Shrewsbury, N. Radiography revealed a high level of mineralized bone formation that conformed to the form of the original collagen disc containing LMP-1s transfected marrow cells. No mineralized bone formation was observed in the negative control cells transfected with a reverse-oriented version of the LMP-1s cDNA that did not code for a translated protein , and absorption of the carrier appeared to be well underway. Histology revealed new bone trabeculae lined with osteoblasts in the LMP-1s transfected implants.
Analysis of Proteoglycan Synthesis and Secretion in Cell Culture Systems
No bone was seen along with partial resorption of the carrier in the negative controls. Clone No.
A base-pair fragment was gel purified from that digest. The isolated base-pair and base-pair cDNA fragments were ligated by standard methods to form a fragment of bp. The linearized vector was recovered and ligated to the chimeric base-pair cDNA fragment.
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