I am new to CO-IP.
I am following this protocol for CO-IP
Lysis buffer 1
0,25ml 1M Tris-Cl
0,15ml 5M NaCl
0,5ml 10% NP40
0,25ml 10% Nadeoxycholate
MQ 3,85ml
1/2 pill EDTA free
Lysis buffer 2 (high salt)
250ul 1M Tris-Cl
500ul 5M NaCl
50ul 10% NP40
25ul 10% Nadeoxycholate
MQ 4.2ml
1/2 pill EDTA free
Lysis buffer 3 (low salt)
250ul 1M Tris-Cl
50ul 10% NP40
25ul 10% Nadeoxycholate
MQ 4.75ml
1/2 pill EDTA free
Day 1
1- Resuspend in medium
2- count cells
3- Wash once with cold PBS
4- Add 1ml cold lysisbuffer 1 with EDTA free protease inhbitor.
5- transfer to an eppendorf tube
6- Add 5ul benzonase
7- Incubate 30min @ 4Co in a 50ml tube on a roller bank
8- Pass 3-5x through a blue needle
9- Spin 10 min at 10000g at 4Co
10- Transfer supernatant to a new tube
11- Save 150ul for total lysate
12- Add 850ul to 50ul beads agarose A and add 10ul Ab
13- Spin 16 rpm o/n @ 4oC
Day 2
1- Short spin 20"
2- remove supernatant
3- Wash 1x with lysisbuffer 1
4- Short spin 20"
5- remove supernatant
6- Wash 1x with lysisbuffer 2
7- Short spin 20"
8- remove supernatant
9- Wash 1x with lysisbuffer 3
10- Short spin 20"
11- Remove all wash.
12- Add 150ul elution buffer (0.1M glycine Ph2)
13- Incubate 10' @ RT with rotation (do not exceed 10')
14- Short spin 20"
15- Transfer to a new tube
16- Add 15ul neutralization buffer (Tris HCL ph8.1)
19- Add loading buffer and Boil samples @95oC for 5'
20- Short spin and load on a 4-20% gel
the question is Why I m seeing this massive band along the membrane and how can I optimize the protocol.
thank you in advance
Dear Imen Jridi,
First question: How much protein (microgram) did you load per slot? Similar appearances result from slot overloading
Second question: Were protease-inhibitors included in the lysis buffers? (I assume that the "1/2 pill EDTA free" included protease- and phosphatase inhibitors)
Third question: Is it possible that the target proteins heavily post-translationally modified? This would result in protein species with different (apparent) molecular weigths that together generate these massive bands
Hi,
Thank you Alexander for your reply.
1- I did not determine the concentration of proteins in samples, but I loaded 50ul of each.
2- yes,
3- I don't know about the modification.
However, I did the CO-IP again, I did precleared the beads and after cintrifugation, I took the supernatant and incubated again with beads and antibodies. after elution, i boiled the samples and they became yellow.
would you please explain that for me
Dear Imen Jridi,
The yellow color indicates that the sample was acidic, as bromophenol blue in the loading buffer is a pH indicator that turns yellow under acidic conditions in the sample/loading buffer (which I hope contains freshly added reducing agent such as DTT or bet-mercaptoethanol). The massive bands could then also be a consequence of protein aggregation. Would a high salt elution buffer also work for your target proteins to avoid the acidic pH buffer? Please also make sure that you know the amount of protein that you load on the gel (20 µg is usually more than enough, in particular for IPs) to avoid gel overloading. Use a BCA or DC assay for example, these assays are finished quickly.
Thank you so much. for elution buffer, i m using 0.1M glycine 2Ph and then Neutralization is 1M Tris-HCL ph8. then Input there is no bamd at all so i am thinking may be the antobodies or the beads to pull down the proteins is not working
Why is the protease inhibitor EDTA free? How does it make sure that metal ion dependent proteases are inhibited as well? They could chop down anything including capture antibodies.
Concerning "then Input there is no band at all" - did you check the protein transfer after Western Blot via ponceau to see if protein transfer was successful in general?
To optimize the protocol, I would prepare the beads first by blocking them via resuspension in albumin solution, wash once, then incubate with antibodies, then add to sample. otherwise a lot of unspecific binding might occur.
Are your antibodies monoclonal or polyclonal?