Histone Western Blot
Licia Miller Product Manager
The basic principles and operation steps of Western Blot experiments of histones are similar to those of other conventional proteins, both of which rely on specific antibodies to detect the presence and expression of target proteins. However, due to the special properties and functions of histones in cells, special attention may be required during the experiment.
First, in terms of sample preparation, histones usually need to be extracted from the nucleus, which may require the use of specific nuclear extraction reagents and may require additional steps to purify the histones. Second, the electrophoresis conditions may need to be adjusted because the molecular weight of histones is small, and the concentration of SDS-PAGE electrophoresis may need to be adjusted to ensure good separation.
When choosing antibodies, it is necessary to use antibodies against specific histone modifications (such as acetylation, methylation) that are essential for histone function. In addition, since the expression level of histones may be relatively low, more sensitive detection methods such as chemiluminescence may be needed to improve the sensitivity of detection.
When selecting internal reference proteins, appropriate internal reference proteins, such as Histone H3, should be selected to ensure the accuracy and reproducibility of the results. Finally, in terms of data analysis, specific software may be required to analyze the modification status of histones, which is different from analyzing the expression of conventional proteins.
When performing Western Blot experiments on histones, it is critical to ensure that the experimental conditions are optimized to ensure the accuracy and reliability of the results.
The following histone blotting protocol is commonly used to detect histones derived from purified calf thymus.
Procedure
1. Prepare 0.5 μg of calf thymus or acid-extracted histones per lane and dilute them in 1× LDS sample buffer containing 100 mM DTT.
2. Heat the sample to 95°C for 5 minutes.
3. Briefly centrifuge the sample to recover the sample volume caused by condensation formed in the tube.
NOTE: Please note that protein loading from cell lysate needs to be determined empirically.
4. Prepare a 10% Bis-Tris gel with a thickness of 1.0 mm . It is recommended to use a higher percentage gel ( such as 15%) to more effectively separate histones.
5. Load the histone sample, remembering to include pre-stained protein standards.
6. Place the gel in MES-SDS electrophoresis buffer and run at 200 V for 35 min.
Tip: When working with smaller protein resolution, it is recommended not to allow the dye front to completely flow out of the gel.
7. For protein transfer in gel, refer to the instructions provided with the transfer device. The specific instructions will vary depending on the transfer method used (i.e., semi-dry blotting or wet blotting).
Tip: Generally, transfer times between 30 and 90 minutes should be sufficient for efficient protein transfer; using 1× transfer buffer/20% methanol for 30 Transfer at 4°C for 70 min.
Note: For optimal retention of histones, a nitrocellulose membrane with a pore size of 0.2 mm is recommended.
8. Verify that histone transfer was successful and loading was equal using Ponceau staining. Dilute Ponceau stain with dH2O.
9. Block the membrane with 5% BSA/0.1% TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 hour at room temperature (RT).
10. If desired, cut the membrane into strips and prepare primary antibody at dilution recommended on the datasheet in blocking buffer (5% BSA / 0.1% TBST) .
11. Add blocking peptide (1 μg/ml) as needed and incubate at room temperature for 20 minutes on a rotating platform.
12. Incubate the membrane with the primary antibody for 1.5 hours at room temperature or overnight at 4°C.
13. Rinse the membrane briefly with 0.1% TBST, then wash twice with the same buffer for 5 minutes each , followed by two 10-minute washes.
14. Dilute the secondary antibody with 1% BSA/0.1% TBST solution as recommended by the official website, and then incubate the membrane with the secondary antibody at room temperature for 1 hour [e.g. Ab176443, goat anti-rabbit IgG H&L (HRP) polyclonal antibody].
15. Wash the membrane twice with 0.1% TBST for 5 minutes each, and then wash twice more for 10 minutes each.
16. Perform ECL, ECF, or IR detection according to the manufacturer's instructions, e.g., add ECL reagent for 3 min at room temperature, and then capture WB images using different exposure times: 10 s, 30 s, 1 min, 2 min, 3 min, 4 min, and 5 min.
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