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.
Amended on 1 February 2019
I must sadly and repeatedly say that ELISA method is neither quantitative nor specific method at all.
I must recommend that utilization of new proteomics PDMD method, which is quantitative and specific method (please see file; HepG2 Fucoidan).
I have searched for Nuclear factor NF-kappa-B p105 subunit/DNA-binding factor KBF1/Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1/NF-kB among my human protein database made from PDMD method in vain. This seems due to the differentiation/development difference.
By the way, Nuclear factor-related proteins are present as follows in the freshly prepared cell and tissue homgenates; i.e.,
Fetal hepatocyte Hc has Nuclear protein ZAP3/YLP motif-containing protein 1 (581-1951) at 5.7, and hepatoma HepG2 (15y, male) has Nuclear receptor coactivator 1/Renal carcinoma antigen NY-REN-52 (92-1441) at 4.8 μg/mg of cell protein, respectively.
Liver tissue of LC (with leprosy; female) has Nuclear autoantigen Sp-100 /Speckled 100 kDa (401-879) at 0.86, LC (named as No.6; male) has Nuclear receptor subfamily 4 group A member 1/Early response protein NAK1/TR3 orphan receptor (151-598) at 4.4, and HCC (No.6) has Nuclear pore complex protein Nup214/214 KD Nucleoporin/CAN protein (275-2090) at 10.1 μg/mg of tissue protein, respectively.
Serum of 3 children's biotin-deficiency patients (all with alopecia);
Serum of 3y girl (with GSD-1b) has Nuclear autoantigenic sperm protein (710-788) at 6.2, serum of 4mo boy has Nucleolar protein 5 (89-529) at 1.8, and serum of 1y girl has Nucleolar complex protein 2 homolog (169-749) at 4.5 μg/mg of serum protein, respectively. Interestingly, these Nuclear factor-related proteins in the patient's bloods have disappeared by the biotin therapy. This may be linked to the disppearance of serum Keratin-associated protein/KAP by the biotin therapy (please see file; JMBT alopecia). It is noteworthy that biotin deficiency does not induce the alopecia in adult patient, and Keratin-associated protein/KAP does also not appear in adult patient's serum.
Serum of light gait disorder patient (22y, female) has Nuclear receptor subfamily 6 group A member 1/Germ cell nuclear factor (350-480) at 4.6, healthy serum of 52y female has Nucleoplasmin-3 (80-178) at 11.6, and healthy serum of 33y male has Nuclear receptor subfamily 4 group A member 2/Orphan nuclear receptor NURR1 (147-598) at 2.8 μg/mg of serum protein, respectively.
Interestingly, Nuclear factor-related proteins seem to be differently expressed between child and adult (differentiation/development differences). Healthy child has not expressed Nuclear factor-related proteins, but biotin deficient child has expressed them. Healthy adult seems to express them, however severe gait disorder patient (adult) does not express them.
We have recently reported that Connectin/titin, Albumin and Human serum biotinidase are the markers of adult or the protein showing the differentiation/development differences (please see files; JMBT alopecia and The Fascio effect). Apolipoprotein B-100 expression seems to be under the control of virus.
Furthermore, I have searched for Protease inhibitors/PIs among my protein database, and have found that cultured cells and cancer tissues seem to contain lower PIs.
Protease inhibitors/PIs are significantly lower in the Cancer tissues than the Normal tissues (p = 0.05, single-sided test; Mann-Whitney's U test n1 = 3, n2 = 3).
Thus, protein purification from cultured cells and cancer tissues may be more difficult than normal organs.
Cancer tissues;
HCC tissue (with PBC) has Beta-Casein (180-226) at 0.77 μg/mg of tissue protein.
HCC tissue (named as No.6) has Alpha-2-Macroglobulin/Alpha-2-M/A2M (1220-1474) at 0.19 μg/mg of tissue protein.
Hepatoma HepG2 (cultured without fucoidan) has Metalloproteinase inhibitor 4/Tissue inhibitor of metalloproteinases 4/TIMP-4 (122-224) at 0.85 μg/mg of cell protein.
Normal tissues;
LC tissue (named as No.6) has Amyloid beta A4 protein/Alzheimer's disease amyloid A4 protein/PN-II (722-770) at 0.25, Heparin cofactor II/HC-II (230-499) at 1.7, and Plasma serine protease inhibitor/Plasminogen activator inhibitor 3/PAI-3 (112-406) at 1.6 μg/mg of tissue protein, respectively. Total PIs becomes to be 3.55 μg/mg of tissue protein.
LC tissue (with leprosy) has Cystatin S (100-141) at 0.49, Heparin cofactor II/HC-II/HLS2 (382-499) at 1.3, Kallman syndrome protein/KAL1/Anosmin-1 (334-680) at 3.9, and Metalloproteinase inhibitor 1/TIMP-1/EPA (56-207) at 1.6, and Orosomucoid/Alpha-1 Acid glycoprotein 1 (64-201) at 1.6 μg/mg of tissue protein, respectively. Total PIs becomes to be 8.89 μg/mg of tissue protein.
Normal liver (with pseudo-liver cancer) has Alpha-2-Antiplasmin/Alpha-2-PI/A2AP (414-491) at 1.2, and Alpha-2-Macroglobulin/Alpha-2-M/A2M (1220-1474) at 0.98 μg/mg of tissue protein, respectively. Total PIs becomes to be 2.18 μg/mg of tissue protein.
Amended on 1 February 2019
I must sadly and repeatedly say that ELISA method is neither quantitative nor specific method at all.
I must recommend that utilization of new proteomics PDMD method, which is quantitative and specific method (please see file; HepG2 Fucoidan).
I have searched for Nuclear factor NF-kappa-B p105 subunit/DNA-binding factor KBF1/Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1/NF-kB among my human protein database made from PDMD method in vain. This seems due to the differentiation/development difference.
By the way, Nuclear factor-related proteins are present as follows in the freshly prepared cell and tissue homgenates; i.e.,
Fetal hepatocyte Hc has Nuclear protein ZAP3/YLP motif-containing protein 1 (581-1951) at 5.7, and hepatoma HepG2 (15y, male) has Nuclear receptor coactivator 1/Renal carcinoma antigen NY-REN-52 (92-1441) at 4.8 μg/mg of cell protein, respectively.
Liver tissue of LC (with leprosy; female) has Nuclear autoantigen Sp-100 /Speckled 100 kDa (401-879) at 0.86, LC (named as No.6; male) has Nuclear receptor subfamily 4 group A member 1/Early response protein NAK1/TR3 orphan receptor (151-598) at 4.4, and HCC (No.6) has Nuclear pore complex protein Nup214/214 KD Nucleoporin/CAN protein (275-2090) at 10.1 μg/mg of tissue protein, respectively.
Serum of 3 children's biotin-deficiency patients (all with alopecia);
Serum of 3y girl (with GSD-1b) has Nuclear autoantigenic sperm protein (710-788) at 6.2, serum of 4mo boy has Nucleolar protein 5 (89-529) at 1.8, and serum of 1y girl has Nucleolar complex protein 2 homolog (169-749) at 4.5 μg/mg of serum protein, respectively. Interestingly, these Nuclear factor-related proteins in the patient's bloods have disappeared by the biotin therapy. This may be linked to the disppearance of serum Keratin-associated protein/KAP by the biotin therapy (please see file; JMBT alopecia). It is noteworthy that biotin deficiency does not induce the alopecia in adult patient, and Keratin-associated protein/KAP does also not appear in adult patient's serum.
Serum of light gait disorder patient (22y, female) has Nuclear receptor subfamily 6 group A member 1/Germ cell nuclear factor (350-480) at 4.6, healthy serum of 52y female has Nucleoplasmin-3 (80-178) at 11.6, and healthy serum of 33y male has Nuclear receptor subfamily 4 group A member 2/Orphan nuclear receptor NURR1 (147-598) at 2.8 μg/mg of serum protein, respectively.
Interestingly, Nuclear factor-related proteins seem to be differently expressed between child and adult (differentiation/development differences). Healthy child has not expressed Nuclear factor-related proteins, but biotin deficient child has expressed them. Healthy adult seems to express them, however severe gait disorder patient (adult) does not express them.
We have recently reported that Connectin/titin, Albumin and Human serum biotinidase are the markers of adult or the protein showing the differentiation/development differences (please see files; JMBT alopecia and The Fascio effect). Apolipoprotein B-100 expression seems to be under the control of virus.
Furthermore, I have searched for Protease inhibitors/PIs among my protein database, and have found that cultured cells and cancer tissues seem to contain lower PIs.
Protease inhibitors/PIs are significantly lower in the Cancer tissues than the Normal tissues (p = 0.05, single-sided test; Mann-Whitney's U test n1 = 3, n2 = 3).
Thus, protein purification from cultured cells and cancer tissues may be more difficult than normal organs.
Cancer tissues;
HCC tissue (with PBC) has Beta-Casein (180-226) at 0.77 μg/mg of tissue protein.
HCC tissue (named as No.6) has Alpha-2-Macroglobulin/Alpha-2-M/A2M (1220-1474) at 0.19 μg/mg of tissue protein.
Hepatoma HepG2 (cultured without fucoidan) has Metalloproteinase inhibitor 4/Tissue inhibitor of metalloproteinases 4/TIMP-4 (122-224) at 0.85 μg/mg of cell protein.
Normal tissues;
LC tissue (named as No.6) has Amyloid beta A4 protein/Alzheimer's disease amyloid A4 protein/PN-II (722-770) at 0.25, Heparin cofactor II/HC-II (230-499) at 1.7, and Plasma serine protease inhibitor/Plasminogen activator inhibitor 3/PAI-3 (112-406) at 1.6 μg/mg of tissue protein, respectively. Total PIs becomes to be 3.55 μg/mg of tissue protein.
LC tissue (with leprosy) has Cystatin S (100-141) at 0.49, Heparin cofactor II/HC-II/HLS2 (382-499) at 1.3, Kallman syndrome protein/KAL1/Anosmin-1 (334-680) at 3.9, and Metalloproteinase inhibitor 1/TIMP-1/EPA (56-207) at 1.6, and Orosomucoid/Alpha-1 Acid glycoprotein 1 (64-201) at 1.6 μg/mg of tissue protein, respectively. Total PIs becomes to be 8.89 μg/mg of tissue protein.
Normal liver (with pseudo-liver cancer) has Alpha-2-Antiplasmin/Alpha-2-PI/A2AP (414-491) at 1.2, and Alpha-2-Macroglobulin/Alpha-2-M/A2M (1220-1474) at 0.98 μg/mg of tissue protein, respectively. Total PIs becomes to be 2.18 μg/mg of tissue protein.
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
Why aren't you homogenizing the tissue instead..we do that for our lab and it works fine and the protein yield is quite good with that.
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