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Anti-xxxxxxxxx

D313-3
Page 1
For Research Use Only.
Not for use in diagnostic procedures.

CLONALITY

QUANTITY
IMMUNOGEN
KLH conjugated synthetic peptide, CKHIQpSNLDFpSPVNS FORMURATION
PBS containing 50% Glycerol (pH 7.2). No preservative is contained. This antibody solution is stable for one year from the date of purchase when stored at -20°C.
APPLICATIONS-CONFIRMED
2 g/mL for chemiluminescence detection system 2 g/800 L of cell extract from 8 x 106 cells
SPECIES CROSS REACTIVITY on WB


Entrez Gene ID

REFERENCES

1) Kasahara, K., et al., EMBO J. 29, 2802-2812 (2010) [WB, IC]
2) Chen, M. S., et al., Mol. Cell Biol. 23, 7488-7497 (2003)
3) Jin, J., et al., Genes Dev. 17, 3062-3074 (2003)
4) Jiang, K., et al., J. Biol. Chem. 278, 25207-25217 (2003)
5) Zou, L. and Elledge, S. J., Science 300, 1542-1548 (2003)
6) Zhao, H. and Piwnica-Worms, H., Mol. Cell Biol. 21, 4129-4139 (2001)

For more information, please visit our web site https://ruo.mbl.co.jp/
MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.
URL https://ruo.mbl.co.jp/
e-mail support@mbl.co.jp, TEL 052-238-1904
D313-3
Page 2
SDS-PAGE & Western blotting
1) Wash cells 3 times with PBS and suspends them in Laemmli’s sample buffer, then sonicate briefly (up to 10 sec.). Adjust 2) Boil the samples for 3 min. and centrifuge. Load 20 L of the sample per lane in a 1-mm-thick SDS-polyacrylamide gel 3) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hr. in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacturer's manual for precise transfer procedure. 4) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for overnight at 4°C. 5) Wash the membrane with PBS-T (0.05% Tween-20 in PBS) (5 min. x 3 times). 6) Incubate the membrane with primary antibody diluted with 1% skimmed milk (in PBS, pH 7.2) as suggested in the APPLICATIONS for 1 hr. at room temperature. (The concentration of antibody will depend on the conditions.)
7) Wash the membrane with PBS-T (5 min. x 3 times). 8) Incubate the membrane with the 1:10,000 anti-IgG (Rat)-HRP (MBL; code no. IM-0825) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hr. at room temperature. 9) Wash the membrane with PBS-T (5 min. x 3 times). 10) Wipe excess buffer on the membrane, and then incubate it with appropriate chemiluminescence reagent for 1 min. Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 11) Expose to an X-ray film in a dark room for 15 min. Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting; UV irradiated HeLa and hydroxyurea treated HeLa) Western blot analysis of Phospho-Chk1 (Ser296)
Lane 1: HeLa, UV irradiated (30 J/m2, 30 min.) Lane 2: HeLa, 3 mM Hydroxyurea treated (3 hr.) Lane 3: HeLa, untreated D313-3
Page 3


P

Immunoprecipitation
a
1) Wash 1 x 107 cells 2 times wit g
h PBS and resuspend them with 1 mL of ice-cold Extraction buffer (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1% NP-40, 2 e
mM EDTA, 1 mM EGTA, 50 mM -glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 50 mM sodium pyrophosphate, 1 mM PMSF), then sonicate briefly (up to 10 sec.) and incubate for 30 min. on ice. 3
2) Centrifuge the tube at 12,000 x g for 5 min. at 4°C and transfer the supernatant to another tube. 3) Mix 20 L of 50% protein G sepharose beads slurry resuspended in 250 L of Extraction buffer with primary antibody as suggested in the APPLICATIONS. Incubate with gently agitation for 1 hr. at room temperature.
4) Wash the beads 3 times with 0.2 mL of Extraction buffer. 5) Add 800 L of cell lysate (prepared sample from step 2)), then incubate with gentle agitation for 1 hr. at room temperature. 6) Wash the beads 6 times with 0.2 mL of Extraction buffer. 7) Resuspend the beads in 20 L of Laemmli’s sample buffer, boil for 3 min. and centrifuge. 8) Load 10 L of the sample per lane in a 1-mm-thick SDS-polyacrylamide gel (12.5% acrylamide) for electrophoresis. 9) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hr. in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacturer's manual for precise transfer procedure. 10) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for overnight at 4°C. 11) Incubate the membrane with primary antibody diluted with 1% skimmed milk (in PBS, pH 7.2) as suggested in the APPLICATIONS for 1 hr. at room temperature. (The concentration of antibody will depend on the conditions.)
12) Wash the membrane with PBS-T (0.05% Tween-20 in PBS) (5 min. x 3 times). 13) Incubate the membrane with the 1:10,000 anti-IgG (Rat)-HRP (Jackson ImmunoResearch; code no. 112-035-175) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hr. at room temperature. 14) Wash the membrane with PBS-T (5 min. x 3 times). 15) Wipe excess buffer on the membrane, and then incubate it with appropriate chemiluminescence reagent for 1 min. 16) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 17) Expose to an X-ray film in a dark room for 15 min. Develop the film as usual. The condition for exposure and development (Positive control for Immunoprecipitation; hydroxyurea treated HeLa) Phospho-Chk1
Immunoprecipitation of Phospho-Chk1 (Ser296)
Lane 1: HeLa, untreated, whole cell lysate (1 x 105 cells/lane) Lane 2: HeLa, 3 mM Hydroxyurea treated (3 hr.), whole cell lysate (1 x 105 cells/lane) Lane 3: HeLa, untreated, whole cell lysate (0.5 x 105 cells/lane) Lane 4: HeLa, 3 mM Hydroxyurea treated (3 hr.), whole cell lysate (0.5 x 105 cells/lane) Lane 5: IP from untreated HeLa with D313-3 Lane 6: IP from 3 mM Hydroxyurea treated (3 hr.) HeLa with D313-3 Lane 7: IP from untreated HeLa with isotype control (MBL; code no. M090-3) Lane 8: IP from 3 mM Hydroxyurea treated (3 hr.) HeLa with isotype control (M090-3) D313-3
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Immunocytochemistry
1) Spread the cells on a glass slide, then incubate in a CO2 incubator for one night.
2) Remove the culture supernatant by careful aspiration.
3) Fix the cells by immersing the slide in 3.7% paraformaldehyde (PFA)/PBS for 20 min. at room temperature (20~25oC). 4) Wash the slide 2 times in PBS for 5 min. 5) Immerse the slide in 0.1% Triton X-100/PBS for 10 min. at room temperature. 6) Wash the slide 2 times in PBS for 5 min. 7) Add blocking buffer (5% normal donkey serum /PBS) to the cell and incubate for 20 min. at room temperature. 8) Add 200 L of the primary antibody diluted with blocking buffer as suggested in the APPLICATIONS onto the cells and
incubate for 1 hr. at room temperature. (Optimization of antibody concentration or incubation condition is recommended if 9) Wash the slide 3 times in 0.1% Triton X-100/PBS for 5 min. 10) Add 100 L of 1:1,000 anti-IgG (Rat)-Alexa Fluor®488 (Invitrogen; code no. A11006) diluted with blocking buffer onto the cells. Incubate for 30 min. at room temperature. Keep out light by aluminum foil. 11) Wash the slide 3 times in 0.1% Triton X-100/PBS for 5 min. 12) Wipe excess liquid from slide but take care not to touch the cells. Never leave the cells to dry. 13) Counter stain with DAPI for 5 min. at room temperature. 14) Wash the slide 2 times in PBS for 5 min. 15) Promptly add mounting medium onto the slide, then put a cover slip on it. (Positive control for Immunocytochemistry; UV irradiated HeLa) HeLa, UV irradiated
HeLa, untreated
(30 J/m2, 30 min.)
Immunocytochemical detection of Phospho-Chk1 (Ser296) in HeLa
These data were kindly provided by Dr. Kousuke Kasahara. (Division of Biochemistry, Aichi Cancer Center Research Institute)

Source: http://www.mbl.co.jp/e/ruo/pdf/D313-3.pdf

Working paper no 3

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