We sought to find a co-IP condition that minimized non-specific binding, but retained the interaction between Oct4 and Nanog, using F9 embryonal carcinoma cells as our source material. Under a mild buffer condition (Condition 1), Oct4 was successfully immunoprecipitated but Nanog was not (), indicating disruption of the Oct4-Nanog interaction during the preparation of cell extract or performance of IP. When the F9 cell extract prepared under Condition 1 was applied to a gel filtration column, Oct4 (38 kDa) was eluted in fractions No. 12, 13 and 14 (corresponding to a molecular mass in the range of 60-400 kDa) while the majority of Nanog (34 kDa) was eluted in fractions No. 8 through 11 (larger than 400 kDa) (). The separation of Oct4 and Nanog was consistent with our failure to co-IP these proteins under Condition 1. The results from gel filtration also suggest that Oct4 and Nanog likely had already dissociated before IP, probably during the making of cell extract.
Comparison of different buffer conditions for co-IP of Oct4 and Nanog
Gel filtration analysis of the Oct4-Nanog interaction
To preserve the Oct4-Nanog interaction, the same procedure was carried out under a milder buffer condition, using Buffer B for making cell extract and performing IP (Condition 2). Buffer B has a lower concentration of NP-40 and lacks Triton X-100. This condition still failed to co-IP Nanog, suggesting that the interaction between Oct4 and Nanog is unstable (). In addition, numerous non-specifically co-precipitated proteins were observed in a silver-stained gel due to the mildness of Condition 2 (). At this point we considered cross-linking the cellular proteins before extraction to preserve the Oct4-Nanog complex, but were faced with the problem that cross-linking likely would also increase co-IP of non-specifically bound proteins. To address this dilemma, we tried to decrease non-specific binding by using a denaturing buffer (Buffer C, also known as RIPA buffer) containing two anionic detergents (sodium deoxycholate and SDS) in addition to a higher concentration of NP-40 () for making cell extract and performing IP. Before performing the IP using cross-linking, we decided to test Buffer C alone to see how effective it was at removing non-specific protein-protein interactions (Condition 3). Given the strong denaturing effect of this buffer, we expected to see a substantial reduction in the amount of non-specific binding; however, we were surprised to find that there was only a slight difference between Buffers A and C (). As expected, Nanog was not co-precipitated with Oct4 using Buffer C (). Although co-IP was not successful, the Oct4 signal was stronger for IP performed using Buffer C compared with those done using Buffers A or B when immunoprecipitated proteins from these three conditions were run in the same Western blot, developed onto a single X-ray film (, arrows). This result could be due to the increased accessibility of epitopes under the denaturing condition provided by Buffer C.
In our next experiment, Condition 4, we sought to maintain the Oct4-Nanog interaction by cross-linking proteins with DSP. Silver staining of a protein gel displayed several bands of proteins that seemed to have been specifically co-precipitated with the Oct4 antibody (, arrows). Consistent with this result, Nanog was also co-precipitated (). Accordant with the co-IP result, Oct4 and Nanog were eluted in the same fractions (mainly in Nos. 12 and 14) in gel filtration column chromatography of the cell extract prepared under this condition (). Under denaturing conditions such as this, the unfolding of proteins makes size estimation by gel filtration chromatography inaccurate. Finally, we tested whether the use of a different cross-linking reagent would alter the results. This time, formaldehyde was used to cross-link proteins instead of DSP, utilizing Buffer C (Condition 5). Unexpectedly, under this condition Nanog was not observed to be co-immunoprecipitated, suggesting that the Oct4-Nanog interaction was not preserved by cross-linking with formaldehyde. This result could be explained by the fact that formaldehyde cross-links molecules at a shorter distance (approximately 2Å) than DSP does (12 Å) [11
Our results highlight the instability of the interaction between Oct4 and Nanog in F9 cell extract and demonstrate the effectiveness of using DSP to maintain this interaction. Furthermore, the detection of several proteins that were co-precipitated with the Oct4 antibody under this condition indicates that DSP potentially preserved the binding of other partners in this complex. More detailed studies of these proteins are required to confirm the interactions of these proteins with Oct4 and Nanog, as well as the functional meaning of the interactions. Our results assume increased importance when one considers them in light of recent findings by Yamanaka’s group [13
]. They have shown that transduced Oct4, in combination with three other transcription factors, can convert mouse fibroblast cells into ES-like cells. The possibility that Oct4 could be used exogenously for stem cell therapy makes a deeper understanding of Oct4’s interactions within the cell all the more pressing for the advancement of stem cell biology.