6543min8+0。 Cell lysate was prepared 3 minutes before sampling (now ready). RIPA: protease inhibitor: phosphatase inhibitor =99: 1: 1. 6-well plate protein collection needs 200ul per well, and the dosage is calculated. Taking the six-hole protein as an example, RIPA 1200ul, protease inhibitor 12ul and phosphatase inhibitor 12ul are added in proportion, and put on ice after mixing. (The reagent RIPA lysate (middle), protease inhibitor mixture (100×) and protease inhibitor mixture (100×) I used were all purchased from * * * Company).
2. Discard the old culture medium and wash it with PBS for three times.
3. Add 200ul of freshly prepared lysis solution to each well, immediately put it on ice and lyse it on an oscillator for 30 minutes.
(Always operating on ice)
1. Take out animal tissues (brain, lung, etc.). ) and divided into three or more parts (one WB, one qPCR and one standby), and immediately stored at -80℃ after taking out. (Note: Take a piece of tissue and freeze it at -80℃ immediately. Repeated freezing of samples will affect the protein to be tested. It is best to do the experiment with fresh samples. )
2. Prepare cell lysate (for later use), the tissue: RIPA= 1mg: 10ul, which can be adjusted according to the actual situation. RIPA: protease inhibitor: phosphatase inhibitor =99: 1: 1. Mix and put on ice.
3. Cut off a part of tissue about 90mg and put it into a vibration homogenizer with steel balls for homogenization. The cover or core of this homogenizer is usually placed at -20℃ and should be taken out when in use. The operation should be fast, and the temperature can basically be maintained at 1min.
4. Add the homogenate to 90 microliters of the prepared cracking solution and crack it on an ice shaker for 30 minutes.
I used BCA protein quantitative kit from * * * company to determine the concentration of extracted protein, which is slightly different from the instructions, as follows:
1. Dilute BSA standard solution to make a standard curve: take 7 EP tubes, add 30ul of normal saline to each tube, add 30ul of BSA to the first tube, mix well, then take 30 ul to the second tube, and dilute it several times in turn, and the last tube will not be blank (i.e. background value). The concentrations of the eight standards are 2ug/ul, 1ug/ul, 0.5ug/ul, 0.25ug/ul, 0. 125ug/ul, 0.0625ug/ul and 0.03 125ug/ul respectively.
Can be prepared in advance:
Wear a mask and gloves, select a glass plate of 1.0mm, carefully scrub the glass plate with a tube brush and detergent, and rinse it with clean water. After the clamping groove is aligned and clamped, put it on the soft rubber gasket, clamp it, and fill the plate with distilled water to test whether the plate leaks. If there is no water leakage after observing for several minutes, pour out the water in the board and hang it upside down to dry.
1. Prepare 8 ml 10% separation gel, add 3.04 ml distilled water, 2.7 ml 30% acrylamide, 1.5M? Ph 8.8±2.0 ml, 10% sodium dodecyl sulfate 80 microliters, 10% alkaline phosphatase 80 microliters, TEMED 3.2ul microliters. After adding TEMED and mixing, pour the glue immediately, and gently glue the glass plate from left to right with 1ml gun head to avoid uneven glue concentration and bubbles. After adding, replace the 200ml gun head, and add distilled water more carefully from left to right to seal it to avoid breaking the newly poured separation glue. Let it stand. When a horizontal broken line appears between water and glue, it will solidify.
2. After the separation glue is solidified, pour out the distilled water sealed with water, and put the filter paper strip in the corner of the glass plate to absorb water to speed up the residual water flow out and invert it. Prepare 4 ml of 5% separation gel, add 2.7 ml of distilled water, 670 ml of 30% acrylamide and 0.5M? 500 ul of pH8.8 500ul of10% SDS, 40 ul of10% AP and 4 ul of TEMED. After adding temed and mixing, immediately pour the glue. As above, gently add thick glue from left to right with a 1ml gun head, rinse it with a comb with a hole of 10, and gently horizontally insert it into the thick glue to stand and solidify. The remaining concentrated glue can be added to the glass plate along the comb to make it completely sealed.
3. At this time, the pre-packaged boiled protein samples, pre-dyed protein markers and a small amount of loading buffer can be melted at 4℃.
cheap/little trick
1.AP and TEMED are neurotoxic and carcinogenic substances, so be careful when using them.
2. When pouring the glue, the line of sight should be flush with the glue surface. Pay attention to the operation to avoid the glue splashing into your eyes.
This kind of glue can be cured in half an hour at room temperature. If it is cold or needs rapid curing, it can be placed in an incubator at 37℃ 15min or so.
4. If you run the glue immediately the next morning, in order not to delay the lunch time, you can adjust the glue the night before, take off the glass plate after solidification, put it in distilled water with a clip and comb, and keep it at 4℃ overnight.
1. Clamp the glass plate in the electrophoresis tank. First, put the prepared 1× electrorheological fluid into the inner tank. After ten minutes, observe whether there is any leakage. If there is leakage, readjust the glass plate and clamp it again until there is no leakage. Otherwise, the electrorheological fluid may leak out before the glue is used up.
2. Gently pull out the comb, add 5ul of pre-dyed protein marker into the first hole, add 20ul of protein samples to be detected in turn into the second to seventh holes, and add 10ul pre-melted loading buffer into the eighth hole. When adding the sample, add the sample gently, and be careful not to add the sample to other holes, or add the sample too quickly to cause the sample to overflow.
3. Insert the positive and negative poles, and pay attention to check that the positive and negative poles cannot be inserted backwards. Adjust the voltage to 80V (thick gel) and run for 0.5h to adjust the voltage to 100V (gel separation). After about 1.5h, maker separated obviously, and a blue buffer band appeared at the bottom of the gel. At this point, electrophoresis is complete. After use, make a good registration in the manual of the experimental instrument.
Note: Avoid blue bands from running out of the separation gel, so the target protein may also run out, so stop electrophoresis when neat and horizontal blue bands appear at the bottom of the separation gel.
cheap/little trick
1. After the positive and negative electrodes are inserted into electrophoresis, small bubbles will rise at the bottom of the electrophoresis tank, and the number and rising speed of bubbles are proportional to the voltage. If the bubble is different from the normal time and remains the same after a few minutes of observation, it is necessary to check whether the electrophoresis solution is added wrongly or the electrophoresis solution is mixed wrongly.
2. Generally, the concentration gel is 80V 0.5h, and the separation gel is100 ~120v1~1.5h. The specific gel operation voltage and time are related to the size of the target protein, experimental instruments and laboratory environment, and the best conditions need to be explored many times. If the target protein is small, shorten the running time as much as possible, and observe the position of maker in time to prevent the target protein from running out. I have experimented with 80V 0.5h, 80V 1h, 120V 1.5h, 120V 1h, 10V 1.5h,/kloc-0. 1 10V 1h。 The results showed that the separation effect of target protein and internal standard was the best under the conditions of concentrated gel 80V 0.5h and separation gel10 v1.5 h. Under this condition, I repeated several experiments and the results were consistent. Therefore, the optimum electrophoresis conditions of this target protein in our laboratory were summarized.
3. Electrophoresis directly affects the development of the target protein, especially the dimer of the target protein. If the electrophoresis conditions are optimized, the results can clearly show protein expression. Therefore, it is a key step to explore the best electrophoresis conditions.
4. After electrophoresis, the filter paper and PVDF membrane needed for membrane transfer can be prepared and soaked in the electro-transfer liquid. It is suggested that after the first WB, the cardboard with different sizes should be cut open and marked with area and number of holes (i.e. number of samples) so that it can be used at any time after storage.
5.5 It is best to clamp PVDF membrane with flat-headed tweezers. WB and clamping the upper left corner of the membrane can minimize the damage to the protein on the membrane.
After electrophoresis, take out the glass plate, rinse it slightly, clean the electrophoresis tank and put it away. Gently pry open the glass plate, and cut the glue on the glass plate according to the position of the marker and the molecular weight of the target protein. In order to prevent cutting deviation, the cut cardboard with the same size as the glue can be placed under the glass plate. Generally, two small pieces of glue, target protein and internal reference, need to be cut from a piece of glue, and the cut glue should be soaked in electrorheological fluid respectively.
1. According to the size of each glue cut, cut out 6 pieces of filter paper and 1 piece of PVDF membrane with the same size and soak them in electrorheological fluid. The prepared cardboard can be used as template scissors. PVDF membrane was activated with methanol for 20s, and then soaked in electrorheological fluid.
2. Make a "sandwich" transfer film on the electrometer, put three layers of filter paper at the bottom, then put PVDF film, glue and three layers of filter paper in turn, and gently drive away bubbles with a glass rod (be careful not to touch the upper and lower layers of filter paper, otherwise it will easily lead to short circuit). Cover, adjust the current according to the total area of the membrane, and start the machine for 2 hours.
3. At the same time of electric rotation, the following liquids can be prepared, and the required volume can be calculated in advance according to the number of membranes:
Take a piece of membrane as an example, seal 5ml+ dilute 4ml of primary antibody+dilute 4ml of secondary antibody, and prepare 15ml if necessary.
5% BSA solution (w/v): prepare 15ml 5% BSA solution (w/v), weigh 0.75g BSA crystal, dissolve it in 15mlTBST, mix it evenly with a vortex shaker, prepare it now, and store it at 4℃ (if the target protein is phosphorylated protein).
5% milk (w/v): prepare 15ml 5% milk (w/v), weigh 0.75g skimmed milk powder, dissolve it in 15ml TBST, mix it with a vortex shaker, prepare it now, and store it at 4℃.
1×TBST lotion: When in use, dilute 100×TBST to 1× with double distilled water and store it at room temperature.
cheap/little trick
1. Some electro-rotating membranes use NC membranes. I haven't tried NC membrane, but the WB results of NC membrane used for many times in the laboratory next door are still not satisfactory, and it has been improved after switching to PVDF membrane. Therefore, PVDF membrane is recommended. You can buy Millipore's PVDF membrane. A roll is a little expensive, but it can be used in the laboratory for several years. It is best not to buy sub-packaged PVDF films from dealers. In another laboratory, a classmate thought it was cheap, and after only a few times, he only bought the 100cm2 membrane from the reagent company. As a result, the price is too high and the film is still fake. Protein samples were not wasted until the end of the experiment. Therefore, it is recommended to buy original PVDF membrane from Millipore Company.
2. As for the electro-rotation mode, some adopt wet rotation and some adopt semi-dry rotation, and both modes are acceptable. Personally, I think it is very convenient and simple to change from semi-dry to dry.
After electrotransformation of 1., the membrane was cut according to the position of the marker, the size of the target protein and the internal reference. You can cut a small corner in the upper left corner of the side marked maker to let everyone know which belt is the 1 sample.
2. The target protein and internal reference were sealed with 5% BSA and 5% milk, respectively, and incubated on an oscillator for 2 hours at room temperature. According to the size of the box, the amount of sealing agent added should not penetrate through the film.
1. Prepare primary antibody p-STAT 1 (company), dilute it to 1:500, add more than 4 ml of 5% BSA, then add 8 ul of primary antibody P-STAT 1, mix well and prepare now.
Prepare the first actin antibody (company), dilute it with 1:2000, add 4ml of 5% milk prepared above, then add 2ul of the first actin antibody, and mix well. Now it is ready for use.
2. Take the PVDF membrane out of the sealing solution, carefully put it into the fingers of plastic gloves with flat-headed tweezers, add the primary antibody, seal it, make the membrane completely soaked in the primary antibody, and put it in the refrigerator at 4℃ overnight. I usually make a small box with a sealed membrane, put it in a large glass plate, put PVDF membrane in it, add an antibody to the membrane from top to bottom with a gun, completely immerse it, then fix it on a shaker with a PCR sampler, and incubate at 4℃ overnight.
The next day, the primary antibody was recovered, marked, stored at -20℃ and reused for 3~4 times. Put the membrane into 1×TBST, and wash the supernatant three times on a shaker, each time for 10 minute.
1. Prepare secondary antibody (anti-mouse or anti-rabbit) with species specificity, dilute it with 1:2000, add 4ml of 5% milk prepared above, then add 2ul of secondary antibody, mix well and prepare now.
After 2.2. TBST washed the membrane, added the second antibody and incubated for 2 hours at room temperature on a shaker.
1. The secondary antibody was recovered after incubation and stored at -20℃. Wash the membrane on the 1×TBST oscillator for three times, each time for 10 minute.
2. protein of one item, that is, one film needs 100ul luminescent liquid A and 100ul luminescent liquid B. Calculate the dosage according to the number of films, and mix luminescent liquids A and B at 4℃ in advance.
3. Add the mixed luminous liquid into the darkroom, cut a small corner in the upper left corner of the film (consistent with PVDF film), and expose the film for different durations, such as 15s, 30s, 1min, 5min, etc. Then develop in developer and fixer and put it in tap water. After leaving the darkroom, hang the film to dry.
You can also take pictures with Bio-rad gel imaging system and choose different exposure time for imaging.
Trick 1. It is necessary to explore the best exposure time of protein through different exposure times, which can only be adopted after repeated experiments. I develop in the darkroom first, and then take pictures with Bio-rad gel imaging system. Comparing the results, I found that the results behind were clearer and the background was cleaner. Since then, I have been taking pictures with the imager.
2. Clamp the membrane with ordinary tweezers, but only the upper left corner of the membrane, so as not to damage the exposed protein band on the membrane.
3. Be sure to cut a small corner or make other marks on the producer's side in the upper left corner of the film, so as to clearly correspond to each sample after development.
4. If exposed in a dark room, a bright band may appear just after adding the internal reference protein luminescent liquid, and the longest exposure of 15s is enough.
If the development effect is not good, the film can be regenerated and re-developed, which saves the steps of glue preparation, electrophoresis and electric conversion.
1. Add 4ml of regenerated liquid of western blotting membrane and shake the bed at room temperature for 30 minutes.