13 replies to this topic

[alex2815]

C:\Users\Archea\Downloads\13.jpgI am having a problem with Tie-2 western blots. There is only on strong band showed up around 250 kDa, whereas Tie-s should be 140 kDa. I also have high background and each lane shows strong signal. see attached picture.

Wet-transferred at 100V for 1.5h, with 10% Methanol, block with 5% dry milk in TBST for 1h; primary ab dilution, 1:500, overnight incubation @ 4^。C;   2^nd Ab, 1:10000 dilution, 1h @ R.T.

Attached Thumbnails

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[alex2815]

What is the problem that cause these black lanes? I used standard washing method: three times washing, each for 5-10mins.

[science noob]

Maybe it's the antibody problem.  Try the same antibody from a different company.  Some produce alot of non-specific binding.  There seems to be a band at the 100-150kDa region in some of the lanes.

How long did you block the membrane in 5% milk? And what membrane were you using - nitrocellulose or PVDF?

Edited by science noob, 15 December 2011 - 04:58 PM.

[alex2815]

science noob, on 15 December 2011 - 04:56 PM, said:

Maybe it's the antibody problem.  Try the same antibody from a different company.  Some produce alot of non-specific binding.  There seems to be a band at the 100-150kDa region in some of the lanes.

How long did you block the membrane in 5% milk? And what membrane were you using - nitrocellulose or PVDF?

I used nitrocellulose membrane and blocked it for 1h @ RT.

[science noob]

Have you used housekeeping antibodies before? (tubulin, actin, GAPDH)

If yes, try to run one which you got nice staining from (low background).  This can definitely confirm an antibody problem.  Or try repeating the blot (same conditions) with another antibody which worked nicely prior.

[mdfenko]

you may want to try diluting the primary more. also, what is the diluent? you should dilute with blocking agent in the solution.

[alex2815]

science noob, on 15 December 2011 - 11:53 PM, said:

Have you used housekeeping antibodies before? (tubulin, actin, GAPDH)

If yes, try to run one which you got nice staining from (low background).  This can definitely confirm an antibody problem.  Or try repeating the blot (same conditions) with another antibody which worked nicely prior.

Yes, I just tried actins and it turned out great. The 250kDa bands are still there, but they are weak.

[alex2815]

mdfenko, on 16 December 2011 - 07:40 AM, said:

you may want to try diluting the primary more. also, what is the diluent? you should dilute with blocking agent in the solution.

I used 5% dry milk to dilute primary and secondary Ab.

[alex2815]

science noob, on 15 December 2011 - 11:53 PM, said:

Have you used housekeeping antibodies before? (tubulin, actin, GAPDH)

If yes, try to run one which you got nice staining from (low background).  This can definitely confirm an antibody problem.  Or try repeating the blot (same conditions) with another antibody which worked nicely prior.

Attached picture is the b-actin film. What do you think? Can you be sure that tie-2 primary Ab caused this problem? Why are the 250 kDa bands still there? I stripped the membrane for 10min at least, but not over 15min, and then standard washing process.

[science noob]

alex2815, on 16 December 2011 - 11:51 AM, said:

science noob, on 15 December 2011 - 11:53 PM, said:

Have you used housekeeping antibodies before? (tubulin, actin, GAPDH)

If yes, try to run one which you got nice staining from (low background).  This can definitely confirm an antibody problem.  Or try repeating the blot (same conditions) with another antibody which worked nicely prior.

Attached picture is the b-actin film. What do you think? Can you be sure that tie-2 primary Ab caused this problem? Why are the 250 kDa bands still there? I stripped the membrane for 10min at least, but not over 15min, and then standard washing process.

So was this the same blot as the one you attached earlier post-stripping? The protein is definitely there and transferred well.  But you need to make sure your Tie-2 is the correct size and not 250 kDa.  Try running a few samples on a fresh membrane and stain for b-actin. You should see a clear band minus the 250kDa.

I think it is the primary problem.  Try antibodies from another company.  Make sure they have a different immunogen (read the spec sheet) because some antibodies from different companies can be similar.

[alex2815]

science noob, on 17 December 2011 - 01:33 AM, said:

alex2815, on 16 December 2011 - 11:51 AM, said:

science noob, on 15 December 2011 - 11:53 PM, said:

Have you used housekeeping antibodies before? (tubulin, actin, GAPDH) If yes, try to run one which you got nice staining from (low background). This can definitely confirm an antibody problem. Or try repeating the blot (same conditions) with another antibody which worked nicely prior.

Attached picture is the b-actin film. What do you think? Can you be sure that tie-2 primary Ab caused this problem? Why are the 250 kDa bands still there? I stripped the membrane for 10min at least, but not over 15min, and then standard washing process.

So was this the same blot as the one you attached earlier post-stripping? The protein is definitely there and transferred well. But you need to make sure your Tie-2 is the correct size and not 250 kDa. Try running a few samples on a fresh membrane and stain for b-actin. You should see a clear band minus the 250kDa. I think it is the primary problem. Try antibodies from another company. Make sure they have a different immunogen (read the spec sheet) because some antibodies from different companies can be similar.

This is the same membrane as i posted earlier. After stripping and incubation with b-actins, the 250 kDa bands are still there. Why? On the first film, nothing or very faint bands between 100-150 kDa. Tie-2 is about 140 kDa, not 250.

Thank you a lot!

[science noob]

alex2815, on 17 December 2011 - 12:18 PM, said:

science noob, on 17 December 2011 - 01:33 AM, said:

alex2815, on 16 December 2011 - 11:51 AM, said:

science noob, on 15 December 2011 - 11:53 PM, said:

Have you used housekeeping antibodies before? (tubulin, actin, GAPDH) If yes, try to run one which you got nice staining from (low background). This can definitely confirm an antibody problem. Or try repeating the blot (same conditions) with another antibody which worked nicely prior.

Attached picture is the b-actin film. What do you think? Can you be sure that tie-2 primary Ab caused this problem? Why are the 250 kDa bands still there? I stripped the membrane for 10min at least, but not over 15min, and then standard washing process.

So was this the same blot as the one you attached earlier post-stripping? The protein is definitely there and transferred well. But you need to make sure your Tie-2 is the correct size and not 250 kDa. Try running a few samples on a fresh membrane and stain for b-actin. You should see a clear band minus the 250kDa. I think it is the primary problem. Try antibodies from another company. Make sure they have a different immunogen (read the spec sheet) because some antibodies from different companies can be similar.

This is the same membrane as i posted earlier. After stripping and incubation with b-actins, the 250 kDa bands are still there. Why? On the first film, nothing or very faint bands between 100-150 kDa. Tie-2 is about 140 kDa, not 250.

Thank you a lot!

I still do think it's the primary antibody problem.  Maybe try a higher dilution? 1:500 can be quite high for certain primaries.  Try 1:1000.  Did the spec sheet recommend 1:500?

Edited by science noob, 17 December 2011 - 07:13 PM.

[alex2815]

Data sheet recommends 1:200 as start dilution (range from 1:100 to 1: 1000). I used that dilution at the beginning and the film was even worse, dirty and cloudy, then I tried 1: 500 on new membrane which is the film i uploaded at first. Higher dilution might help to reduce the nonspecific binding, but the problem is that with further dilution(1: 1000), the right bands (140kDa) are not likely gonna show up. The other people in my lab do not think the problem is from the primary but from transfer, blocking or washing, even if it is an old antibody.

Anyway, thank you very much for your advise. I will try one more time next week and keep my fingers crossed...

science noob, on 17 December 2011 - 07:13 PM, said:

alex2815, on 17 December 2011 - 12:18 PM, said:

science noob, on 17 December 2011 - 01:33 AM, said:

alex2815, on 16 December 2011 - 11:51 AM, said:

science noob, on 15 December 2011 - 11:53 PM, said:

Have you used housekeeping antibodies before? (tubulin, actin, GAPDH) If yes, try to run one which you got nice staining from (low background). This can definitely confirm an antibody problem. Or try repeating the blot (same conditions) with another antibody which worked nicely prior.

Attached picture is the b-actin film. What do you think? Can you be sure that tie-2 primary Ab caused this problem? Why are the 250 kDa bands still there? I stripped the membrane for 10min at least, but not over 15min, and then standard washing process.

So was this the same blot as the one you attached earlier post-stripping? The protein is definitely there and transferred well. But you need to make sure your Tie-2 is the correct size and not 250 kDa. Try running a few samples on a fresh membrane and stain for b-actin. You should see a clear band minus the 250kDa. I think it is the primary problem. Try antibodies from another company. Make sure they have a different immunogen (read the spec sheet) because some antibodies from different companies can be similar.

This is the same membrane as i posted earlier. After stripping and incubation with b-actins, the 250 kDa bands are still there. Why? On the first film, nothing or very faint bands between 100-150 kDa. Tie-2 is about 140 kDa, not 250.

Thank you a lot!

I still do think it's the primary antibody problem.  Maybe try a higher dilution? 1:500 can be quite high for certain primaries.  Try 1:1000.  Did the spec sheet recommend 1:500?

[alex2815]

I have a question. When using higher concentration of primary Ab and Secondary Ab the results showed many non-specific bands but the bands we want were faint or not showed up, is it gonna help to make the interested bands more clear and visible and reduce non-specific binding if decrease the concentration of primary Ab and Secondary Ab?

science noob, on 15 December 2011 - 04:56 PM, said:

Maybe it's the antibody problem.  Try the same antibody from a different company.  Some produce alot of non-specific binding.  There seems to be a band at the 100-150kDa region in some of the lanes.

How long did you block the membrane in 5% milk? And what membrane were you using - nitrocellulose or PVDF?