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Ebook Lehninger Biokimia Astri Aldelina. d_c01_ 10/27/03 AM Page 1 mac76 mac_reb: chapter 1 THE FOUNDATIONS OF. The International Journal of Biochemistry & Cell Biology 41 () – Contents lists available at ScienceDirect The International Journal of. Download as PDF · Download Biokimia 1 / Aisjah Girindra Download as Postscript. Print Biokimia 1 / Aisjah Girindra Send to Email Biokimia 1 / Aisjah Girindra.


Buku Biokimia Pdf

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Proteins were visual- were suspended in the same but nominally calcium free solution ized by the colloidal Coomassie staining according to the Anderson CaCl2 replaced with 0.

[2.3.1.55 Deleted entry. kanamycin 60-N-acetyltransferase identical to EC 2.3.1.82 aminoglycoside

The time resolution of the measurements was 1 s. The supernatant was mixed serum for 30 min.

At the beginning the separation was con- ondary antibody Alexa Fluor , Donkey Anti-Rabbit IgG diluted ducted slowly at 50 V for 30 min to 1 h until the samples entered for 2 h, at room temperature. Then the voltage was increased to V. Cell immo- for 0. Images were subjected gel. Proteins were visualized with the colloidal Coomassie staining to background-subtraction and deconvolution, using Huygens 3. Western blot analysis range of pixel-intesities.

The primary antibodies used were grp- tion reagent Amersham Pharmacia Biotech. Cells were then washed in sodium dodecyl sulfate-polyacrylamide gel and proteins were elec- PBS, and secondary antibodies Alexa Fluor , Goat Anti—Mouse troblotted onto nitrocellulose membranes.

Image acquisition was K. This setup thus allowed the resolution of nm in the xy, and nm in the z direction. For details of our protocol see localization Section 2 and Supplementary Figure 1. Moreover, a slightly acidic medium ca.

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Such treatment of cells of divalent cations chelators in buffers, and pH of sucrose gradi- resulted also in accumulation of large STIM1-immunoreactive gran- ent was adjusted to 6.

Notably, the specimen was Figure 2 differs from others, but some of protein bands of the PAM have counterparts in the ER and mitochondria.

As it is shown in Fig. These data corre- spond well to results published by Lillemeier and co-workers, and could be explained by the presence of plasma membrane proteins forming clusters attached to cytoskeleton.

B Translocation Chen and co-workers Chen et al. Using this isolation procedure two bands of STIM1 have been always observed. The lower band corresponds to the STIM1 which is deglycosylated and the upper to the glycosylated form.

Incubation of the collected material with 7 M urea and 2 M thio-urea resulted in an dissociation of the high purity PM. Immunoblot analysis of protein components of subcellular fractions pre- pared from Jurkat cells. Representative blots showing the relative amounts of STIM1 is present in different protein complexes characteristic protein markers in isolated subcellular fractions are shown. Depletion of the ER tary Fig. This might indicate that SOCE activation, the rearrangement of STIM1-containing protein the plasma membrane and mitochondria form stabile structures complexes were analyzed.

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This technique allows talk between these two structures e. The typical pat- tern of the total cellular proteins obtained by sequential use of 3. The results obtained indicate that the established Zhang et al.

This protein interacts with Orai1 protein located in the PM, eling of protein complexes. Particularly, stimulation of cells by thapsigargin the PAM fraction, an intracellular localization of these proteins was resulted in the increase of proportion of STIM1 localized in the investigated. We estimated the relative amount of STIM1 and Orai1 complexes of relatively higher molecular mass than in control cells in selected cellular fractions.

As shown in Fig. Subcellular localization of STIM1 protein. A Subcellular fractions were prepared as is described in Material and methods. A typical result of immunoblots are shown. For ORAi1 presented values are means from two independent experiments.

A typical results of immunoblot are shown. Presented values are means from two independent experiments. Results presented in Fig.

Furthermore, we also found that the tion of the SOCE pathway have been published so far. To acquire Z-series of images described in control and stimulated cells, suggesting that forma- of the mitochondrial marker grp75 present in the mitochondrial tion of direct contacts between selected regions of different cellular matrix and the OMM surface Szabadkai et al.

As shown on Fig. PM in both control and Tg treated cells, without any gross change In the present study we analyzed interactions between the ER in their structure. In other words signals Fig.

We observed changes in the mitochondrial contact sites Rizzuto et al. Two different experimental approaches were chondrial, ER and PM markers, coalescing into dynamic protein applied for separation of selected subcellular fractions, including complexes comprising all three organelles.

PM and PAM. B and C — a typical results of an immunoblot from two separate experiments shown. The second one and inner mitochondrial membranes are involved in the PAM for- based on a biotinylation of the intact cell surface proteins and iso- mation. The detection of the only selected mitochondrial and ER lation of biotinylated PM subfractions from cellular homogenate. It was suggested that, mitochondria located in the vicinity As we shown in Fig.

It was pro- al. ANT chondria to the plasma membrane Quintana et al. This observation is in agreement with in the ER fraction suggests that the composition of PAM fraction is the data presented by Quintana and co-workers, who pointed out unique and not accidental. They found that the plasma membrane centration in the cytosol reached plateau Quintana et al.

Thus, it probably was to short period for mito- protein composition of the PAM fraction is the same as or different chondrial translocation to be observed. In control cells and during then the subfraction of the ER in the proximity to the plasma mem- early phase of SOCE mitochondria form a partially continuous net- brane Pichler et al. In contrary to these data our results work in the narrow space between the nucleus and the PM.

Analysis of the subcellular fractions isolated from Jurkat cells Peirce et al. A 3D structure of mitochondria in resting state and upon capacitative calcium entry in Jurkat cells. Mitochondria are shown in red, STIM1 in green. It is proteins. Lebiedzinska et use of PAM fraction isolated from Jurkat cells correspond well to the al. Detection of STIM1 in the higher molecular mass complexes concomitantly with its decreased par- Acknowledgments ticipation in the low molecular mass complexes indicates serious changes in proteins interrelationships upon stimulation of cells by This work was supported in part by Ministry of Science and thapsigargin.

This work was also partly supported Fig. Supplementary data tacts is newly formed and the rest comes from already preexisting ones. Recently it has been reported that close apposi- the online version, at doi These struc- et al. The same authors characterized protein composition of PAM.

Moreover a possible function of these structures related to phospholipids Abbreviations: PAM, plasma membrane associated membranes; MAM, mito- particularly phosphatidylserine and phosphatidylinositol synthe- chondria associated membranes; ER, endoplasmic reticulum; SOCE, store-operated sis and transportation within a cell was also considered.

More calcium entry; CCE, capacitative calcium entry. The distance smaller than 30 nm between 1 mM MgCl2 , 0. Recently postu- CCE. After next 3 min the cells were used for subfractionation. The homogenate was tion.

Crude membrane fraction containing Achleitner et al. Formation of functional intermembrane plasma membrane, mitochondria and PAM fraction was suspended structures between the ER and the mitochondria was demon- in 5 mM Bis—Tris, 0.

The distance between interacting mem- made on the base of the solution: Interestingly, close contacts between Tris—HCl pH 7. Crude mitochondrial fraction was All isolated Co-isolation of ER, mitochondria and PM suggests that dynamic subcellular fractions were suspended in mM mannitol, 75 mM associations between these membranes could be fairly tight. The sucrose, 0. More- 2. It may be concluded that PAM are important intermembrane Crude plasma membrane PM-PAM enriched fraction was iso- structures, of which the formation and rearrangement, following lated as described previously Pichler et al.

Rockford, US was used. Materials and methods was incubated with 2. Than cells 2.

Materi Biokimia

MgCl2 pH 7. The eluted proteins membranes for 30 min with Tris-buffered saline TBS Proteins were visual- were suspended in the same but nominally calcium free solution ized by the colloidal Coomassie staining according to the Anderson CaCl2 replaced with 0.

The time resolution of the measurements was 1 s. The supernatant was mixed serum for 30 min. At the beginning the separation was con- ondary antibody Alexa Fluor , Donkey Anti-Rabbit IgG diluted ducted slowly at 50 V for 30 min to 1 h until the samples entered 1: Then the voltage was increased to V. Cell immo- for 0.

Images were subjected gel.

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Proteins were visualized with the colloidal Coomassie staining to background-subtraction and deconvolution, using Huygens 3. Western blot analysis range of pixel-intesities. A group of cells was washed with KRB contain- mouse monoclonal, 1: The primary antibodies used were grp- tion reagent Amersham Pharmacia Biotech. Samples containing 75 goat polyclonal, Santa Cruz, 1: Cells were then washed in sodium dodecyl sulfate-polyacrylamide gel and proteins were elec- PBS, and secondary antibodies Alexa Fluor , Goat Anti—Mouse troblotted onto nitrocellulose membranes.

This setup thus allowed the resolution of nm in the xy, and nm in the z direction. Four subcellular fractions: For details of our protocol see localization Section 2 and Supplementary Figure 1. Moreover, a slightly acidic medium ca. Such treatment of cells of divalent cations chelators in buffers, and pH of sucrose gradi- resulted also in accumulation of large STIM1-immunoreactive gran- ent was adjusted to 6.

Notably, the specimen was Figure 2 differs from others, but some of protein bands of the PAM have counterparts in the ER and mitochondria. As it is shown in Fig. These data corre- spond well to results published by Lillemeier and co-workers, and could be explained by the presence of plasma membrane proteins forming clusters attached to cytoskeleton. B Translocation Chen and co-workers Chen et al.

Using this isolation procedure two bands of STIM1 have been always observed. The lower band corresponds to the STIM1 which is deglycosylated and the upper to the glycosylated form. Incubation of the collected material with 7 M urea and 2 M thio-urea resulted in an dissociation of the high purity PM. Immunoblot analysis of protein components of subcellular fractions pre- pared from Jurkat cells.

Representative blots showing the relative amounts of STIM1 is present in different protein complexes characteristic protein markers in isolated subcellular fractions are shown. ER mark- ers: PM markers: Mitochondrial markers: Depletion of the ER tary Fig. This might indicate that SOCE activation, the rearrangement of STIM1-containing protein the plasma membrane and mitochondria form stabile structures complexes were analyzed.

This technique allows talk between these two structures e. The typical pat- tern of the total cellular proteins obtained by sequential use of 3. The results obtained indicate that the established Zhang et al.

This protein interacts with Orai1 protein located in the PM, eling of protein complexes. Particularly, stimulation of cells by thapsigargin the PAM fraction, an intracellular localization of these proteins was resulted in the increase of proportion of STIM1 localized in the investigated.

We estimated the relative amount of STIM1 and Orai1 complexes of relatively higher molecular mass than in control cells in selected cellular fractions. As shown in Fig. Subcellular localization of STIM1 protein. A Subcellular fractions were prepared as is described in Material and methods.

A typical result of immunoblots are shown. For ORAi1 presented values are means from two independent experiments. A typical results of immunoblot are shown. Presented values are means from two independent experiments. Results presented in Fig. Furthermore, we also found that the tion of the SOCE pathway have been published so far. To acquire Z-series of images described in control and stimulated cells, suggesting that forma- of the mitochondrial marker grp75 present in the mitochondrial tion of direct contacts between selected regions of different cellular matrix and the OMM surface Szabadkai et al.

As shown on Fig. PM in both control and Tg treated cells, without any gross change In the present study we analyzed interactions between the ER in their structure. In other words signals Fig. We observed changes in the mitochondrial contact sites Rizzuto et al.

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Two different experimental approaches were chondrial, ER and PM markers, coalescing into dynamic protein applied for separation of selected subcellular fractions, including complexes comprising all three organelles.Cell ;— Cell Calcium ; Such treatment of cells of divalent cations chelators in buffers, and pH of sucrose gradi- resulted also in accumulation of large STIM1-immunoreactive gran- ent was adjusted to 6.

J Biol Chem ; The elementary unit of store-operated Vance JE.

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