Western blotting materials

Analyze the eluted material by SDS-PAGE or by Western blot Dialyze against a large volume of PBS (no azide) overnight at 4°C. 

Fig. 6. (A) Caspase-7 forms generated by caspase cleavage Pro, pro-region LSU, large subunit and SSU, small subunit. (B) Western blot of cell extracts. Extracts from Jurkat cells incubated with STS for the indicated times and then for 1 h with bzVKD-fmk and STS were subjected to Western blotting with anti-caspase-7 LSU (Anti-LSU), anti-caspase-7 SSU (Anti-SSU), or HRP-streptavidin (HRP-S A). (C) Western blots of captured polypeptides. After incubation with STS for the indicated times and then 1 h with bzVKD-fmk and STS cells were extracted as described in Methods for capture on 6-well dishes coated with caspase-7 capture antibody. Captured material was solubilized in SDS sample buffer and subjected to Western blotting with anti-caspase-7 LSU (Anti-casp-7 LSU), anti-caspase-7 SSU (Anti-casp-7 SSU), or HRP-streptavidin (HRP-SA).

Specificity of the active caspase-7 ELISA was determined by analyzing captured polypeptides. Capture antibody is the caspase-7-specific antibody coated in the ELISA 96-well plates and is different from the anti-caspase-7 antibodies used for western blotting. Polypeptides that are bound by the capture antibody are referred to as captured. Jurkat cells were incubated with STS for 0-4 h and then with bzVKD-fmk for an additional 1 h. Cell extracts were incubated in 6-well plates coated with caspase-7 capture antibody. After washing, polypeptides captured on the plate were solubilized in SDS sample buffer and Western-blotted. Captured polypeptides were blotted with anti-caspase-7 LSU and anti-caspase-7 SSU to detect polypeptides derived from caspase-7. Captured polypeptides were blotted with HRP-streptavidin to detect the polypeptides covalently modified with the bzVKD-fmk inhibitor. Captured polypeptides covalently modified with bzVKD-fmk is the material that the ELISA quantifies. 

Western blotting of protein extracts of various C. roseus plant materials after SDS-PAGE, with polyclonal antibodies raised against the purified enzyme from elicited C roseus cells, revealed several bands. During the development of seedlings, first an immunoresponsive 54.8-kDa band, which is devoid of activity, is noted. Subsequently, a transient increase of a 55-kDa band is observed, which coincides with the occurrence of TDC activity. With the decrease of the TDC activity some other bands could be detected (40, 44, and 67 kDa). In leaves, the 54.8-kDa band could not be detected (178). In a series of in vitro experiments, Fernandez et al. (186) studied the stability of TDC. The presence of Mn, or particularly has a stabilizing... 

At the same time a 14-3-3 protein ELISA was developed, which achieved a relative quantification of the 14-3-3-protein concentration in CS fluid from CJD and non-CJD patients. To determine the sensitivity of the ELISA, the 14-3-3 content in extracts from cattle was also measured in parallel using ELISA and immunoblots. For the ELISA the lowest amount of 14-3-3 protein detectable was a concentration of 0.5ngml and for the Western blot immunoassay a concentration of 4 ng ml This test should make it possible, through its high sensitivity and quantification of the 14-3-3 concentration in investigated material, to distinguish between various... 

Fig. 2. Western blot analysis of protein bound to immunoabsorbant columns. PyBHK EF-2 preparations containing cellular ADP-ribosyltransferase activity were chromatographed on Sepharose 4B coupled to Pseudomonas toxin A antibody or pyBHK ADP-ribosyltransferase antibody. After washing unbound material from the resin with PBS, the bound protein was eluted from the columns with 1 M propionic acid, concentrated under vacuum and subjected to electrophoresis on a 7.5% SDS-polyacrylamide slab gel. In addition. Pseudomonas toxin A standards were subjected to electrophoresis on the gel. Following electroblotting of the proteins from the ge) to a nitrocellulose sheet, the sheet was sectioned and reacted with Pseudomonas toxin A antibody (A-Q or with pyBHK ADP-ribosyltransferase antibody iP-F). Pseudomonas toxin A (A) protein from Pseudomonas toxin A antibody coupled immunoabsorbant B) protein from pyBHK ADP-ribosyltransferase antibody coupled immunoabsorbant (C, D) protein from Pseudomonas toxin A antibody-coupled immunoabsorbant (i ) Pseudomonas toxin A F). The numbers represent Mj. X 10" of the mol.wt. standards...

Fig. 3. Immunoblots of subcellular fractions of CHO and CALU-1 cells. In summary, cells (5 X 10 ) peimeabilized with 1% NP-40 (NP) were treated at 0° C with 250 pg/ml of DNase I and ase A for 2 hr (RD), extracted for 1 hr with 2M NaCl buffer (S), and centrifuged to yield insoluble material (P). The different subcellular fractions (NP,RD,S,P), re senting 100% of cellular protein content, were dissolved in a 4 M urea SDS sample buffer and subjected to western blot analyses. Poly(ADP-ribose) polymerase from CHO and CALU-1 cells were revealed in these cases with C-2-10 and C-1-9 antibodies respectively. Purified calf thymus enzyme (E) is used as reference.

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