The National High-tech Research and Development Program (863 Program) and the National Key Basic Research and Development Program (973 Program, including major scientific research programs) are relatively high-end research projects of the country.

But if you want to get one of them, you need to accumulate enough.

Of course, if you can get one, you can also make people famous, millions of project funds are a piece of cake, and tens of millions of scientific research funds are also very common.

And the start-up capital for such a project is more than 100 times the youth fund that Yuan Weihong has won so far.

When I was about to say anything more, the patient who had finished the examination and follow-up outside had already pushed the door open.

Yuan Weihong stopped talking, and Fang Ziye also hurriedly began to collect his own knowledge points, read and examine the film step by step, accurately diagnose and use medicine, and then got a knowledge point of 0.9.

I was happy.

Because even Zhongnan Hospital is not the kind of incurable and miscellaneous diseases everywhere, there are naturally many incurable diseases, but there are also simple diseases, and patients will not consider wasting resources when registering.

After the outpatient clinic, Fang Ziye got about 15 points of knowledge again, and this time, even if he added some knotting to level 3, he gave a gift to the teacher.

The balance of knowledge points has accumulated to as much as 60 all of a sudden, and at the same time, you can also add a level 3 skill, tonight, Fang Ziye still has cell experiments, which are about to be harvested, and when the time comes, the observation results will definitely be a lot of knowledge points.

"Master, I'll put the white coat first, and then I'll go to the laboratory!"

"Okay, WB run well." Yuan Weihong confessed, as if he was hinting to Fang Ziye, and he knew what Fang Ziye was doing.

Fang Ziye nodded and quickened his pace.

Doing experiments, making money, publishing articles, learning points, fulfilling and happy.

WB is an abbreviation, and Western blot is a method of transferring proteins to a membrane and then using antibodies to detect them.

The ultimate result of a gene expression is the production of the corresponding protein (or enzyme). The HK2 studied by Fang Ziye is actually the first rate-limiting enzyme hexokinase in the biochemical glycolysis process, and HK2 is the abbreviation of hexokinase, which is the enzyme that catalyzes the phosphorylation of hexose, which is the first enzyme of the glycolysis pathway and the absolute rate-limiting enzyme of the glycolysis pathway.

As we all know, glycolysis is the first step in the conversion of sugar into energy, without the first step of glycolysis, it is almost impossible for glucose and fructose to be utilized normally, and if you rely on bypasses such as ketone bodies as the substrate for aerobic oxidation, it will definitely not help.

Therefore, the quantitative detection of the content of HK2 in the absolute amount after overexpression and silencing of bone giant cell tumor is a quantitative evidence for the detection of the role of HK2 in the development of bone giant cell tumor.

Western blot uses polyacrylamide gel electrophoresis, with the analyte being a protein, the "probe" being an antibody, and the "chromogenic" being labeled with a secondary antibody.

The protein samples separated by PAGE are transferred to a solid support (e.g., nitrocellulose film), which adsorbs the protein in a non-covalent bond form and maintains the type of peptide separated by electrophoresis and its biological activity. The protein or peptide on the solid phase carrier is used as the antigen, and the corresponding antibody is immunologically reacted, and then reacted with the enzyme or isotope-labeled secondary antibody, and the protein components expressed by the specific target gene separated by electrophoresis are detected by substrate chromochromization or autoradiography......

The principle sounds quite complicated, but in fact, when it comes to the actual operation, the main steps are mainly the following steps.

Reagent preparation, which is the runway for proteins to run, requires polyacrylamide gel electrophoresis to provide.

Then there are the athletes, who lyse the tumor cells so that the proteins inside are fully released, and then make a standard curve.

Once the standard curve is made, you can start hitting the track with the 'athletes' produced by the cells you're working on.

This is followed by a series of electrophoresis, mold transversion, immune reaction, development, etc., and through special antibodies and reagents, the film of the target protein can be displayed.

The film is scanned or photographed, and the molecular weight and net optical density values of the target band are analyzed using a gel image processing system......

This process is complex, relatively long, and requires more patience.

However, Fang Ziye had already gotten out of the outpatient clinic anyway, and when he came to the laboratory, he did it step by step. And there is also Luo Tingzhu next to him to help a little.

After the results were finally presented, Luo Tingzhu was overjoyed: "Senior Brother Fang, your strip display today is very clear, the last time Senior Brother Fan ran out was dumbbell-shaped." ”

After speaking, she turned her head to look at Fang Ziye's finished film, the strips were clear, there were no miscellaneous bands, and the background was clean!

"There is a problem with the dumbbell-shaped general configuration of the gel, and there is also a possibility that the sample contains too many impurities and is not centrifuged, and then the impurities are deposited in the middle of the wells, and the proteins are naturally pushed to both sides."

"Prepare good glue, resolutely do not use unqualified ones, and don't take risks." Fang Ziye thought about the principle carefully and explained it to Luo Tingzhu.

"And what if there's a strip of trailing?" Luo Tingzhu thought for a while and asked another question, it should be that she had seen such a situation when she followed others.

"If the band is tailed, it is generally because the protein concentration is high or the concentration and time of the primary antibody are too long."

"If the protein content is high, it may be that the literature preparation in the early stage has not been determined, and I think it is most likely because the concentration of the primary antibody is too high and the action time is too long."

"Of course, there are other reasons as well." Fang Ziye looked at his finished film again.

HK2 and β-actin (actin) were internal control bands, and there were three groups: overexpression, normal and silent.

The common GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cannot be used for the internal control of tumor cell experiments, especially those involving the glycolytic pathway.

"You can use this diagram directly with prism, right?" Luo Tingzhu continued to ask.

"It should be about the same, the difference in the thickness and gray scale of the strip is quite obvious. Take a photo, take a photo......" Fang Ziye hurriedly asked Luo Tingzhu to help take a picture.

Luo Tingzhu happily agreed, as agile as a hard-working little squirrel.

At this time, Fang Ziye watched the knowledge points jump rapidly to 134, and suddenly increased the knowledge points by a full seventy points, and he also knew that this WB experiment played a large role in this topic, and it may be a small skeleton that supports the scale of the entire project.

"How's this picture going?" After Luo Tingzhu finished filming, he asked Fang Ziye.

Fang Ziye smiled at his aunt, while Luo Tingzhu thought that his level of taking pictures was not bad, so he clicked a few more.

Then it's time to clean the bench and clean the corresponding equipment.

When everything was over, Fang Ziye suggested: "Senior Sister Luo, it's hard work, let's go have a supper together?" ”

Luo Tingzhu was looking left and right at the photo of WB Chengtu at this moment, tilted his head and nodded: "Okay." ”

……