CHS-003K
Optional Service: Optional requirements on this protein
Applications :
Isolation of intact functional core histones from tissues and cultured cells Protein profiling, post-translational modification and epigenetic analyses Protein profiling of Histones by SDS-PAGE and Western blot
Storage :
Store kit at -20centigrade. Briefly spin small vials prior to opening. Store histones at -20centigrade for up to one week (-80centigrade for longer storage). Avoid multiple freeze thaw cycles. Note: If salt precipitates are seen in the extracts after being frozen, thaw extracts on ice and pipette gently several times until salts are re-dissolved.
Kit Components :
• Lysis Buffer 2 X 100 ml • Extraction Reagent 25 ml • Neutralizing Buffer 10 ml • DTT (1 M) 100 μl
Materials Required but Not Supplied :
Dounce Tissue Homogenizer PBS Tablets PMSF
Preparation :
Store the DTT at -20centigrade. Remaining reagents may be stored at 4centigrade or -20centigrade. Please read the entire protocol before performing the assay. • Neutralization buffer: If a precipitate is visible in the Neutralization Buffer, warm to 37centigrade in a water bath to dissolve it and chill on ice prior to use. Aliquot enough buffer for the number of assays to be performed, and add the appropriate amount of 1 M DTT for a final concentration of 1 mM DTT to the neutralization buffer right before use. • Extraction Reagent, Neutralizing Buffer and DTT (1M): Ready to use. Bring to room temperature (RT) before use.
Separation Protocol :
Total Core Histone Isolation Protocol: 1. Sample Preparation: Wash: Harvest 1 x 10^7 cells and wash twice with ice cold Phosphate Buffered Saline (PBS). Resuspend the pellet in 1 ml of PBS and transfer cells to a 1.5 ml tube. Spin cells at 600 X g for 10 minutes in a microfuge and aspirate supernatant. Cut 100 mg tissue of interest into 2 mm^3 sections and wash twice in a 1.5 ml tube with 1 ml of ice-cold PBS. Centrifuge the cells at 600 x g for 10 min for each wash step and discard the supernatant.
Lysis: Resuspend washed cells in 1 ml of ice-cold Lysis Buffer (optional: containing 2 mM PMSF) and lyse for 10 minutes on ice with intermittent gentle mixing (7-10 tube inversions). Centrifuge the lysate at 600 x g for 10 minutes at 4centigrade. Remove the supernatant and wash the pellet with 0.5 ml of Lysis Buffer. Stain 5 µl of cell lysate with Trypan Blue and view under a microscope at 20x on a glass slide. At least 80-90% of the cells should be lysed. Centrifuge the lysate and discard the supernatant. Repeat the wash step with 0.5 ml of Lysis Buffer and remove supernatant. Resuspend the washed tissue in 1 ml of ice-cold Lysis Buffer (optional: containing 2 mM PMSF) and homogenize it with a Dounce homogenizer on ice to fully disperse the cells. To check for the homogenization efficiency in the tissue sample, view the homogenized sample under a microscope. You should see a uniform suspension. Typically for soft tissues 10 - 15 strokes and for hard tissues 15 - 20 strokes are sufficient. Transfer the lysate into a 1.5 ml tube and incubate on ice for 10 minutes. Spin the minced tissue in a table top microfuge at 600 x g for 10 min. Remove the supernatant and wash with 0.5 ml of Lysis Buffer. Centrifuge as before and discard the supernatant.
Extraction: Completely resuspend pellet in 0.25 ml of ice-cold Extraction Reagent and incubate on ice for 1 hour. Centrifuge at 10000 x g for 10 minutes at 4centigrade and collect the supernatant. Add 0.1 ml ice-cold Neutralizing Buffer containing 1 mM DTT directly to the supernatant and mix well. This isolate contains the core histones. Quantify the histones isolated with any protein quantitation assay. BSA can be used as a standard.
Note: It is possible to scale down or up the sample amount by scaling up or down the volumes used in the protocol with varying yield results.
For Research Use Only. Not intended for any clinical use. No products from Creative BioMart may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative BioMart.
For Research Use Only. Not intended for any clinical use. No products from Creative BioMart may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative BioMart.