It will be OK if you immunoprecipitate for the expressed Flag.

Good luck!

Dear Yulia

are you secreting the protein in the media?

in this case, i think that of you would like ti purify the protein in mg scale with out alter the folding could be better replace the FLAG tag with a c terminal 6 or 10x his tag abd perform an imac purification which works very well for purification from cell culture surnatatnts.

Generally i'm doing it from expi293 which are a powerfull derivate of hek293 able to growth in suspension similar to bacteria up to very high cell density.

you can easly generate the new clobe where replace the FLAG tag with the his just performing a single pcr followed by dpni digestion and trasformation in MACH1 e.coli cells using the PIPE Cloning approach.

You can find details about it on my blog: https://proteocool.blogspot.com/p/cloning.html?m=1

good luck

Manuele

Amended on 4 May 2019

I must sadly declare that Humans has no Dystrophin and/or Microdystrophin at all as assessed by reliable PDMD (Protein-Direct-Microsequencing-Deciphering) method (please see files; HepG2 Fucoidan and JMBT Alopecia). This may due to Species differences (between Humans and Mouse) and to notorious Immunological method (sometimes giving false-positive-binding results).

This sad result is only in accordance with the finding of my teacher famous Late Hon. Prof, Dr. Kunio Yamauchi (The University of Tokyo, Yayoi-cho, Bunkyo-ku, Tokyo, Japan) that humans has no beta-Lactogobulin, alpha-S2 Caseine, and Chymosine/Rennine, but bovine does have. My result is in accordance with his finding; i.e., Insulin and Insulin receptor/IR is present in bovine and porcine, but humans does have not. Therefore, humans has at least 5 small numbers of genes than bovine. Then, notorious Darwin's theory of evolution is surely the false.

Connectin is richly present in adult Human liver tissues, but absent in fetal hepatocyte Hc cells (please see file again; HepG2 Fucoidin). Connectin is also richly present in adult human serum, but absent in children's serum (our unpublished observation). Caldesmon/CDM/Protein CDM is present in fetal hepatocyte Hc (please see file again; HepG2 fucoidin), but absent in adult Human cells.

Actin (originally found in muscle of rabbit, but absent in nematode sperm), Tubulin (originally found in spermatozoids of sea urchin), and Glial fibrillary acidic protein/GFAP are also absent in Humans.

Myoglobin/Mb is absent in Humans, but Humans has Haemoglobin A/HbA. Therefore, I am now condsidering that Human muscle is contracting by utilizing Connectin, Albumin/Alb, and Haemoglobin A/HbA using NADH as an energy source (manuscript in preparation). This estimation is depended the observations of tissues; i.e., perfused liver has Albumin precursor and Haemoglobin A (please see file; SEC column 300A silica), and the major protein of rat thigh muscle is unexpectedly rat Albumin (our unpublished observation). Expectedly fetal hepatocte Hc has alpha-Fetoprotein and does not have Albumin, and hepatoma HepG2 has both alpha-Fetoprotein and Albumin. Albumin may be the adult marker as Connectin is. These reliable data are obtained by quantitative HPLC-Surf-SEC method (please see file; SEC column 300A Silica) and PDMD (protein-direct-microsequencing-deciphering) method.

Adult human serum (without Haemolysis; 22 - 52 y; n = 4) has Albumin at median 392 (range; 324 - 443), Connectin at median 27.6 (range; 16.6 - 38.0), and Haemoglobin A/HbA at median 11.4 (range; 8.5 -13.4) μg/mg serum protein, respectively (my unpublished observation). IgG is 2nd major protein, but Albumin is 1st, Connectin is 3rd, and Haemoglobin A is 4th major serum protein, respectively.

Therefore, I am now condsidering that Human striated muscle is contracting by utilizing Connectin, Albumin/Alb, and Haemoglobin A/HbA using NADH as an energy source. Only this new hypothesis can explain the protein composition of adult human serum.

Then, Human adult livers may produce and provide Connectin, Albumin, and Haemoglobin A to striated muscles. Famous Biochemist Late Dr. Tsuneo Okuyama has shown that NADH dehydrogenase is richly present in membrane of red blood cells (please see file; NADH DHase), and we have shown that the Active transport is energized by NADH (please see file; Ala Transport).

On the other hand, Fibrillin-1, Smoothelin, Stereocilin, Vimentin, TOG array regulator of axonemal microtubules protein 1/Protein KIAA0423/Crescerin-1, Desmuslin/Synemin, Myotilin/Connectin immunoglobulin domain protein, Hornerin, Cytokeratin-5/Keratin, type II/ VK5, Cytokeratin 7/Keratin, type II cytoskeletal 7/VK7, Cytokeratin 15/Keratin, type I cytoskeletal 15, Cytokeratin 2E/Keratin, type II cytoskeletal 2 epidermal, Band 4.1/Erythroid protein 4.1, Dematin/Erythrocyte membrane protein band 4.9, and Neurofilament triplet M protein/Neurofilament medium polypeptide/NF-M are present in Humans.