Peptide Information 3815
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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.
Phospho-HDAC3 (Ser424) Antibody #3815
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. Immunoprecipitation for Native Proteins This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
Protein A Magnetic Beads : Magnetic Separation Rack : or ATP 10 mM for kinase assays : To prepare 0. Preparing Cell Lysates Aspirate media. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS. Remove PBS and add 0. Scrape cells off the plate and transfer to microcentrifuge tubes. Sonicate on ice three times for 5 sec each. The supernatant is the cell lysate. Immunoprecipitation Cell Lysate Pre-Clearing Optional A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads.
Briefly vortex the stock tube to resuspend the magnetic beads. Place the tube in a magnetic separation rack for seconds.
Incubate with rotation for 20 minutes at room temperature. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet. Proceed to immunoprecipitation section. Pre-wash magnetic beads see Cell Lysate Pre-Clearing section, steps 1 and 2. Transfer the lysate and antibody immunocomplex solution to the tube containing the pre-washed magnetic bead pellet.
Incubate with rotation for 20 min at room temperature. Pellet beads using magnetic separation rack. Keep on ice between washes. Proceed to analyze by western immunoblotting or kinase activity section D.
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Sample Analysis Proceed to one of the following specific set of steps. Transfer the supernatant to a new tube. The supernatant is the sample. Analyze sample by western blot see Western Immunoblotting Protocol. Vortex, then microcentrifuge for 30 sec. Transfer supernatant containing phosphorylated substrate to another tube. Immunohistochemistry Paraffin A. Hematoxylin: Hematoxylin Wash sections twice in dH 2 O for 5 minutes each. Staining Wash sections in dH 2 O three times for 5 minutes each. Wash sections in dH 2 O twice for 5 minutes each.
Wash sections in wash buffer for 5 minutes. Prepare ABC solution per manufacturer's recommendations. Remove primary antibody and wash section three times with wash buffer for 5 minutes each. Incubate 30 minutes at room temperature. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
Wash section three times with wash buffer for 5 min each. If desired, counterstain sections with hematoxylin Wash sections in dH 2 O two times for 5 minutes each. Repeat in xylene, incubating sections two times for 10 seconds each. Mount sections with coverslips and mounting medium 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. I am sorry to confirm that we don't have any different lots in stock at the moment.
I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Regrettably, I can suggestthe customer has received a bad vial on this occasion. Therefore, we would be pleased to provide a replacement from the same lot, which will still be covered by our guarantee. Alternatively, I fully understand the customers concerns and if they prefer a credit note in this case, I will be pleased to arrange this for them.
I hope this will be helpful. Please see to the customer answer at the attached file Best regards. Thank you for your message and for kindlyproviding this further information. I am sorry to hear the suggestions made have not improved the results on this occasion. Regrettably, I can suggest you have received a bad vial on this occasion.
I appreciate the time you have spent on these experiments, and would like to offer a refund or credit note in compensation providing the product has been purchased in the last 6 months. In order to arrange this, I would appreciate if you could confirm your order number and date of purchase as previously requested? Thank you for your continued cooperation. Z1 Microscope Zeiss Positive and negative controls used please specify negative control — no primary Optimization attempts problem solving : How many times have you tried the IHC?
Additional Notes Document Attachment: images of the results may be very helpful. If you wish to add it to the complaint form, please attach them to the reply e-mail. Do you obtain the same results every time? ON incubation with ab, cell syncronization Additional Notes. Thank you for taking the time to complete our questionnaire and contact us.
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I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody. The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.
Reviewing this case, I would like to offer some suggestions to help optimise the results. Fixation: To prevent over fixation, whichever method is used, I can suggest this should be for 10 minutes only if this has not already been tried. Please provide further information regarding the permeabilization. I would suggest to try 0. I can recommend to consider reducing the concentration to to reduce the background signal. I would appreciate if you are able to provide an image which would help us to assess the results.
I can suggest to try reducingthe concentration to to reduce the background signal. Incubate overnight 4oC. Could you confirm if the quality of sample been assessed using a loading control? Have the samples reduced and denatured? General: 1. Please confirm the order number and date of purchase. Is the current vial of secondary antibody working well with other primary antibodies?
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I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details. Do you have another batch in stock now? Please give us your suggestion about this matter. He thinks its reproducibility is too weak. Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody.
We do experience occasional batch variation. This often means that it may be necessary for our customers to perform an element of optimisation in their experiments when receiving the antibody. On this occasion I would like to recommend that your customer increases the mass of protein that they are loading on the gel as we typically recommend that ug of protein is loaded.
I would also recommend that the antibody is applied at a dilution of ; as detailed on our datasheet using an overnight incubation. Taken in combination this is very likely to improve the results that your customer has been obtaining significantly. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
Motor protein that translocates PRC1 to the plus ends of interdigitating spindle microtubules during the metaphase to anaphase transition, an essential step for the formation of an organized central spindle midzone and midbody and for successful cytokinesis. May play a role in mitotic chromosomal positioning and bipolar spindle stabilization. Highly expressed in hematopoietic tissues, fetal liver, spleen, thymus and adult thymus and bone marrow.