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View in English? Western blot analysis of cell extracts alone lane 1 , cells expressing Myc tagged to the carboxy terminus of the protein lane 2 and Myc tagged to the amino terminus of the protein lane 3 , using Myc-Tag Antibody. Confocal immunofluorescent analysis of COS cells transfected with a Myc-tagged protein left or untransfected right using Myc-Tag Antibody.
Actin filaments have been labeled with DY phalloidin red. Do not aliquot the antibody. NOTE : Please refer to primary antibody datasheet or product webpage for recommended antibody dilution. From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: Western Blotting Application Solutions Kit.
NOTE : Volumes are for 10 cm x 10 cm cm 2 of membrane; for different sized membranes, adjust volumes accordingly. Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available.
It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment.
This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody. Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our Immunofluorescence Application Solutions Kit.
NOTE : Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips. All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit Methanol , or individually using the catalog numbers listed below.
NOTE : Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation. NOTE : Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, g for minutes will be sufficient to pellet the cells. NOTE : If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation. The antibodies will remain bound to the target of interest during the fixation and permeabilization process.
However, note that some fluorophores including PE and APC are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure. Would you like to visit your country specific website? YES NO. Save This Selection. Citations Image Gallery Learn more about how we get our images.
To Purchase S. Product Size Price. Related Products. Type: All. Citations 1. Citations 7. Citations 2. Dilute to 1X with dH 2 O. Nonfat Dry Milk : Biotinylated Protein Ladder Detection Pack : Blotting Membrane and Paper : This protocol has been optimized for nitrocellulose membranes. Pore size 0. Protein Blotting A general protocol for sample preparation. Treat cells by adding fresh media containing regulator for desired time.
Aspirate media from cultures; wash cells with 1X PBS; aspirate. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice. Sonicate for 10—15 sec to complete cell lysis and shear DNA to reduce sample viscosity. Microcentrifuge for 5 min. Electrotransfer to nitrocellulose membrane Membrane Blocking and Antibody Incubations NOTE : Volumes are for 10 cm x 10 cm cm 2 of membrane; for different sized membranes, adjust volumes accordingly.
Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Wash three times for 5 min each with 15 ml of TBST. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody at and anti-biotin, HRP-linked Antibody at — to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature. Proceed with detection Section D. Mix well. Incubate substrate with membrane for 1 minute, remove excess solution membrane remains wet , wrap in plastic and expose to X-ray film.
Immunofluorescence Immunocytochemistry A. Solutions and Reagents Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our Immunofluorescence Application Solutions Kit NOTE : Prepare solutions with reverse osmosis deionized RODI or equivalent grade water. Adjust pH to 8. Mix well then add 0. NOTE : Formaldehyde is toxic, use only in a fume hood. Allow cells to fix for 15 min at room temperature. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
Proceed with Immunostaining Section C. Block specimen in Blocking Buffer for 60 min. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer. Aspirate blocking solution, apply diluted primary antibody. Rinse three times in 1X PBS for 5 min each. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1—2 hr at room temperature in the dark. For best results, allow mountant to cure overnight at room temperature.
Solutions and Reagents All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit Methanol , or individually using the catalog numbers listed below. Fixation NOTE : Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation. Pellet cells by centrifugation and remove supernatant. Mix well to dissociate pellet and prevent cross-linking of individual cells. Wash by centrifugation with excess 1X PBS.
Discard supernatant in appropriate waste container. Resuspend cells in 0. Proceed to Permeabilization step. Permeabilize for a minimum of 10 min on ice. Aliquot desired number of cells into tubes or wells. Generally, 5x10 5 to 1x10 6 cells per assay. Wash cells by centrifugation in excess 1X PBS to remove methanol.
Repeat if necessary. Incubate for 1 hr at room temperature. Discard supernatant. Incubate for 30 min at room temperature. Protect from light.
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Datasheet Texas Instruments TLC2272CDR