How can I prepare cell lysate from rat brain homogenate for estimating the levels of nuclear factor-kappa B (NF-kB) using ELISA kit? Looking for freezer thaw cycles method in detail.
The success of the lysis method that you propose would in part depend upon the lysis buffer. You may have to use a variety of detergents to extract nuclear proteins successfully. However, as noted by Kou, your method of detection may not allow you to distinguish between the specific activity versus non-specific activity in your freeze-thaw lysate.
Vishwanath Shastry,
We have used HSV-1 infected astrocytes lysates to determine NF-κB p65 protein by ELISA, as well as mRNA levels. Freeze/thaw cycling was found appropriate for the former, and inappropriate for the latter assay. Primary astrocytes were generated from post-natal day 0–1 mouse brains and prepared as described. The astrocytes were washed twice in PBS and treated with 0.25% Trypsin/EDTA for 2 min to dissociate cells. Direct lysis of single-cells ice in either PBS supplemented with 2% BSA or in astrocyte culture medium until subsequent analysis were both tested. Freeze/thaw cycles in 1–4 mg/ml BSA, 50 ng/µl yeast tRNA, 1× RT buffer and water were used. Five hundred astrocytes were lysed, frozen in −80°C and thawed in room temperature 1, 2, 3, or 6 times. Thereafter, the Sandwich ELISA kit was utilized for the measurement of mouse astrocytes' total NF-κB p65. An anti-pan NF-κB p65 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and NF-κB p65 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti- NF-κB p65 (S536) antibody is used to detect phosphorylated NF-κB p65 or biotinylated anti-NF-κB p65 antibody is used to detect NF-κB p65. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG or HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of NF-κB p65(S536) or pan NF-κB p65 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
As already mentioned, we also determined mRNA accessibility after freeze/thaw astrocytes cycles in 1–4 mg/ml BSA, 50 ng/µl yeast tRNA, 1× RT buffer and water. Four genes and four different amounts of freeze/thaw cycles were analyzed per lysis condition. It was concluded that the loss of mRNAs during freeze/thaw cycling is due to self-hydrolysis of nucleic acids, aggregation, and absorption rather than to RNase activity.
Best wishes,
Ilya
Dear Dr Ilya,
Thanks for the detailed response, it is really useful for experiments..
Regards
Vishwanath
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