Protocols

Co-IP Protocol

1. Culture cell, transfection; (avoid cell death; if cells are ok without changing medium after transfection, do not change medium, for example, HEK293 and NSC34.)
2. Aspirate cell culture medium as clean as possible;
3. Add PBS to the cell culture plates to collect cell; (for cell not attached very well, such as HEK293, pipet PBS up and down to detach cell; for attached cell, such as NSC34, using scraper to scrape out the cell.)
4. Transfer the PBS suspended cell into tubes;
5. Centrifuge tubes at 5000rpm or 1500g for 3mins;
6. Aspirate PBS; (aspirate most of the PBS using vacuum, then use pipet tip to remove the rest of the PBS in the tube, avoid losing cells, but also avoid residue of PBS in the tube because the leftover of PBS contains serum and may make gel running looks bad.)
7. Add cell lysis buffer (Pierce IP buffer, Cat #87787, add proteolysis inhibitor) into the tube with cell pallet accordingly; (6-well plate, 100-200ul each well; 6 cm dish, 300-500ul each dish; 10 cm dish, 500-1000ul each dish.)
8. Put the tube on ice for 30 mins;
9. While the waiting for cell lysate, prepare the beads:

• Shake the protein G beads bottle very well to mix the beads and the liquid;
• Take according amount of beads; (for samples from 6-well plates or 6 cm dish, each well/dish sample needs 15ul beads, meaning together with the liquid in the bottle, 30ul needs to be used; for 10 cm dish, each dish needs 20-25ul beads, meaning together with the liquid in the bottle, 40-50ul needs to be used; then calculate the amount of beads needed according to the sample numbers, for example, we have 4 samples from 6 cm dish, then take 120ul out from the protein G beads bottle; DO NOT take beads for each individual sample into individual tube, because beads need to be taken out together and be washed together to avoid treatment difference, which is very easily happen at the beads washing step.)
• Add 1ml PBS to the total beads to wash the beads;
• Do not need to put the tube with beads and PBS onto rotator, just simply rotate the tube up and down by hand for 5-10 times;
• Put the tube into centrifuge and centrifuge at 2000g for 1 min;
• Aspirate the PBS; (aspirate most of the PBS and then use the thinnest pipet tip to remove the rest of PBS.)
• Add cell lysis buffer to the beads and separate the beads into different group; (Pierce IP buffer; if you did not use this cell lysis buffer for your cell lysis, then add the same buffer you used for cell lysis;) Keep in mind we need to divide the beads into separate group as equal as possible, because if there is a subtle difference in the beads amount at the very begining, it is going to affect the result greatly and may not be noticed from the result since heave chain and light chain signal in the final Western Blot result maybe saturated. Also keep in mind that in the end each group of beads will have 500ul of cell lysis buffer, which means if we have 4 samples, finally we will need 2000ul of cell lysis buffer to suspend the beads and aliquot the beads into 4 tubes. However this is hard to achieved because the tube is usually 1.5ml eppendorf tube, so the way I do it is as follows: add 1000ul cell lysis buffer to the beads pallet, mix the beads and the buffer very well by shake the tube, and then take 200ul of the suspended beads for each of the 4 tubes; there will be around of 200ul leftover of suspended beads in the original tube, do not need to worry about this, because I then add another 1000ul into this original tube, mix the beads and the buffer very well and add 200ul of the suspended beads into each of the 4 tubes; now each of the tube have 400ul system and the original tube has around 400ul or less leftover suspended beads; then I add around 300ul cell lysis buffer to the original tube, mix it very well and add 100ul suspended beads into each of the 4 tubes; now each tube has 500ul system and there are some leftover in the original tube, it is ok because it is diluted 3 times and the beads in the original tube is very limited and we can discard it;
• Add IP antibody into each of the tube and rotate in cold room for 30 mins. (Usually 2ug of antibody is used for each IP sample; If I have no idea of the concentration of the antibody, or I am not familiar with the capture ability of the antibody, I just add 2ul of the antibody, that works for most of the commercial antibodies; if I have a lot of beads in each IP, for example I am using 100ul beads to pull down something from four 10 cm dish cell lysate, then I will use around 8ul antibody.)
• After antibody incubation, centrifuge the tube at room temperature, 2000g, 1 min;
• Aspirate the supernatant; (aspirate most of the supernatant first using vacuum, and then use the thinnest pipet tip to remove the residue.)
• Add 1000ul of cell lysis buffer to each tube, rotate up and down by hand for around 10 times, centrifuge at room temperature, 2000g, 1 min;
• Aspirate the supernatant; (same method as mentioned before, aspirate most of the supernatant first using vacuum, and then use the thinnest pipet tip to remove the residue; all the following aspirate steps is done the same way.)
• Close the tube cap to prevent overdry of the beads. (this beads preparation step needs to be coordinate with the cell lysis step very well to prevent the beads wait too long for the cell lysate.)

10. Centrifuge cell lysate tube at >12000g for 7 mins;
11. Collect the supernatant, mark the tube as WCL (whole cell lysate); (avoid take pallet; if the supernatant looks not very clear, put the tube back into centrifuge and centrifuge again for additional time; once the centrifuge is done, immediately take the tube out and avoid waiting because the pallet may get suspended while waiting; do not collect the supernatant near the pallet, for example, if the liquid is 1000ul, just take 900ul-930ul out to avoid collecting pallet, this is very import step to avoid unspecific binding.)
12. Add most of the WCL into beads tube; (DO NOT add all of the WCL into beads, leave around 50-100ul, depending how much WCL you have in total, in the WCL tube; this will be the WCL control for the Western Blot.)
13. After adding WCL into beads, mix well by shaking to make sure beads is suspended well by the WCL; put the tube in cold room rotator to incubate; (incubation time is important and need to be adjusted according to experience; if I have no experience of a particular IP before, then I will start with 3 h incubation time; if in the end Western Blot result shows the unspecific binding is too strong, or I saw some difference between the sample I tried to compare but the difference is not very obvious, then I will try to reduce incubation time; if I was not able to observe the targets that I want to observe, then I will try to extend the incubation time, probably overnight.)
14. Add SDS loading buffer to the WCL sample and boil it immediately and then freeze the sample if Western Blot need to be started the next day;
15. After beads and WCL incubation, centrifuge at 2000g for 1 min;
16. Collect the supernatant into a new tube, label it as flow through and freeze it; (you don’t have to do this, I do it just in case I make any mistake and the sample is too precious; if you don’t want to collect this, just aspirate the supernatant.)
17. Washing beads 3 times, each time rotate the tube in the cold room rotator for 5 min, then centrifuge at 2000g for 1 min, aspirate the supernatant, then do the next round:

• The buffer to be used for the washing step can be very different between different person, and the buffer need to be adjusted according to result; for IP that I have never done before, I will simply start with cell lysis buffer to wash; if in the end I do not see the targets that I want to detect, then I will change the 3^rd step wash into TNT buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1mM DTT, protease inhibitor); if in the end I still do not see the targets that I want to detect, I will use TNT buffer for all the 3 times wash;
• The washing time could also be adjusted according to the result. If after changing the washing buffer, I still do not see the target, I will do 2 times wash, each time 3 min;
• If I see too much unspecific binding, or if I see a difference between groups but the difference is not dramatic, I will extend the washing time to 10 min each; I will also do 5 times wash if needed.

18. After wash, add SDS loading buffer to boil the beads at 100 degree for 5 mins; (the amount the SDS loading buffer to be added is usually 50-70ul; if this sample is from a lot of cell lysate, we could use relatively more SDS buffer, such as 100-120ul.)
19. Centrifuge the boiled beads at 2000g for 1 min;
20. Collect the supernatant, and label it as IP:***.

Western Blot (WB) Protocol

1. Now we have WCL samples and IP:*** samples;
2. Assemble the gel running box, put the pre-made gel in; (after take out the comb of the pre-made gel, carefully use running water to wash the well.)
3. Load sample and marker; (try to establish habits of your own and do all the loading consistently, for example, always load marker on the right side of the samples, because if you make any mistake in the trans-membrane step and you confused yourself which side is which, you will be able to tell from the marker; if you have too many samples to compare, for example, 20 samples, which will not able to fit into one gel because our gel could only load 15 samples for the most, then you can separate the 20 samples into 2 gels, BUT run them for the same amount of time and later transfer these 2 gels onto the same membrane, this will insure that all the following procedures, including blocking, antibody incubation, washing, exposure are exactly the same for all the samples.)
4. Start running the gel at 150 Volts; (you can increase to 180 Volts 15 mins after the gel started to run to save time.)
5. After running, take the gel out, trim the gel to get rid of the parts that has no sample on it; (if I have several gels, I will cut the right top of my gel to mark it as my gel 1 and cut both the right top and right bottom of my gel to mark it as my gel 2 and do not cut anything to mark it as my gel 3; and the reason I cut right side is because right side is always my marker so I won’t be accidently cut the target protein that I want to detect.)
6. Using Invitrogen iBlot Gel transfer stacks nitrocellulose for membrane transfer; Add these five items sequentially: bottom, gel, filter paper, top, and sponge; (before put gel onto bottom, add a little water on the membrane and put gel on, avoid bubble; transfer time is usually 7 mins; if target protein is very big, increase the transfer time to 8-9 mins;) Orientation of the gel when put onto the membrane is very important:

• Always make sure when you look at the membrane, the order of your samples will be the same as you load it; in my case, I will put my gel on and make sure my marker is still on the right side;
• If you have enough space for the gel, try to put the gel looks horizontal to the you; I felt if you put the gel looks vertical to you, the pressure each sample gets might be different; but if you do not have enough space, just ignore this point.

7. Cut the membrane out, put into box and add 5% TBST diluted milk to block the membrane at room temperature for at least 30 mins; (the longer the membrane is blocked, the cleaner the result will be, so you could do blocking overnight; for some antibody, BSA will be recommended for blocking, so for unfamiliar antibody, you will need to check the description of antibody.)
8. After blocking, dilute the blocking milk into 1% milk by TBST and this will be the buffer for further antibody incubation and washing;
9. Dilute primary antibody into 1% milk according to antibody description or experience and incubate the membrane with antibody for at least 1h at room temperature; usually for antibody I have no experience, I will start overnight incubation at cold room; most of the time, longer primary antibody incubation is good, unless in the end the signal you get for the samples you want to compare is saturated and not able to compare, then you want to reduce the primary antibody incubation;
10. Wash membrane after primary antibody incubation:

• 3 times, each time 5 mins, room temperature on shaker;
• DO NOT dump wash buffer directly onto the membrane because if you do so, different parts of the membrane actually gets different extend of wash; if in the end you have to expose the membrane for a very long time, you will be able to notice this difference and that might be the reason sometime the membrane is not evenly black or white.

11. Dilute secondary antibody into 1% milk and incubate the membrane for 1 h at room temperature; (if you are not available 1 hour later, then incubate at cold room for a longer time, although this is not recommended because this will make the final result background high and membrane looks dirty.)
12. Wash membrane after secondary antibody incubation same as step 10;
13. Prepare the Pierce ECL western blotting substrate by 1:1 combination of the two reagents;
14. Put the membrane into TBST to remove the residue of milk;
15. Take the membrane out, put the membrane on tissue paper to let dry for half min, and then put membrane on a SARAN wrap, add ECL substrate onto the membrane; make sure the whole membrane get the substrate; (it might be ok if you just add the substrate to the target size, but then you might miss additional bands that usually happen in Western Blot.)
16. Incubate membrane with ECL for 2 mins and transfer the membrane onto the black plate coming with the ProteinSimple machine;
17. Choose Darkroom-Chemi with Marker-Expose for automatic exposure; you could also change the exposure time manually;
18. Good luck!

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