It is very cold today. I didn't go to bed as late as yesterday, just got up early.

Because I have some final experiments to finish, and then I will say goodbye to the lab for about two months. A little sad, looking back on the dribs and drabs of growing up in the laboratory in the past two months, I have had hard sweat, the distress of failure and the joy of success. ...

It's really sad that I have just mastered some experimental skills and will leave mercilessly. After all, when I come back here, these skills may be returned.

Anyway, this is only a temporary farewell, just to sort out the previous data, and then better design and plan the follow-up experiments.

At the laboratory, we completed the follow-up part of the last wave of immunohistochemistry. Continue to complete WB eluted yesterday (this eluent has a good effect. However, the results have not improved much. A new discovery is the exposure time. This time, I adjusted the exposure time to 600 seconds according to the legend in the manual, and it actually broke out. )

In view of yesterday's question-WB internal reference is used up, but the target gene band is blank, I will answer it today.

1, is the primary resistance confused with the secondary resistance?

A: Combining the results of the two experiments shows that I have not confused the types of primary antibody and secondary antibody.

2. The glue was cut wrong, resulting in the missing target strip?

Answer: there is no problem in cutting the glue, but there is a problem in cutting the internal reference. The molecular weight of the antibody specification is 36KD, and the actual molecular weight is 39KD.

3. Too little tissue protein expression?

A: This is most likely. At present, my WB protein is 30ug, so I can consider further using about 50ug.

4. Because the target protein is a catalytic enzyme, will it lead to protein loss without using related protease inhibitors (such as cokertail)? )?

A: The effect of protease inhibitor PMSF is ok, and other protease inhibitors can be considered in the future if conditions permit.

5. protein (two weeks ago) was stored for too long, and protein was degraded?

A: There may also be this factor, which leads to the reduction of consumption in protein. It is suggested to complete protein extraction as soon as possible.

6. The membrane transfer time (300mA, 2h) is too long, which leads to protein penetrating through the membrane.

A: There are two kinds of target proteins. Today, one protein (64KD) was exposed by me, and the other protein (52KD) has been blank. It is highly suspected that membrane transfer leads to protein loss. Referring to other people's suggestions, the film transfer condition can be set as: 200mA, 1min/1KD.

7. The electrophoresis solution takes too long (I'm too lazy to use someone else's electrophoresis solution-I ran once ten days ago)?

A: Practice shows that the influence is not particularly great, which may affect the uniformity of protein on the membrane.

8. exposure time is too short?

A: For protein with very low expression, it is suggested to extend the exposure time. For example, today, I set the exposure time to 500 seconds and 300 seconds to see the stripes. Although it is weak, I also found the result, which has brought great help to the elimination of the problem.

Add two questions:

9. Is there a problem with the antibody concentration? Maybe it's too low. Can it be improved?

A: I doubled the concentration of 52KD protein, but it was still not exposed. Therefore, for WB white films, the influence of this concentration should be in a relatively backward position.

10, the sensitivity of the exposure liquid?

A: This is one of the key factors. In the second run, under the condition of constant protein content, I controlled the membrane transfer time, prepared fresh antibodies in less than 90 minutes, and used ultra-sensitive ECL exposure solution (only enhanced ECL exposure solution was used for the first time), and the effect was particularly good this time. So I suggest using super-sensitive exposure liquid.

It's time to say goodbye, but I believe this is not forever!