9 replies to this topic

[ehernandez4]

Hi guys

I just did my first western blot a few weeks ago, and i have been trying to troubleshoot ever since. The problem with my blot is that too many bands are seen on the blot and none of them are for my protein of interest the size i was looking for is 42 kDa. I am not sure what the problem could be? not sure if it is my primary antibody that i am using which is monoclonal anti actin in mouse from sigma (clone AC-40), or if its the secondary antibody which is Peroxidase conjugated affinipure goat anti mouse igG form jackson immuno research. If someone could please help me trouble shoot this issue.......

[bob1]

If you can post what you actually did (including solutions, dilutions, wash steps...)  - it will help us troubleshoot, not much to work on in your post above.

[Sotirios]

In order to see if the is your secondary antibody, use it without the first one. I think that the secondary one binds the first. Maybe there is cross reactivity of first antibody or you have only fragments ofrom youw antigen.

[ehernandez4]

bob1, on 15 March 2012 - 12:03 PM, said:

If you can post what you actually did (including solutions, dilutions, wash steps...)  - it will help us troubleshoot, not much to work on in your post above.

Ok, so to begin I was using a TGS running buffer to run the gel for about an hr at 100v,  i am transferring the proteins on a pvdf membrane and I was blocking the membrane for an hour using 5% non fat dry milk in TBS-T for an hour, after the blocking i was directly putting  it  in at a 1:500 primary antibody incubation overnight at 4C with gentle rocking. In the morning i would wash it 3X for 5min with TBS-T, and then proceeded to incubate it with the secondary antibody at a 1:5000 concentration and left it for an hour with gentle rocking. After that i would wash it again 3x with TBS-T for 5 min. After that i would use the Super Signal West Femto Maximum Sensitivity Substrate used a concentration of 1mL total and put it on the membrane incubate for 5 minutes and proceed to image.

the first time i used vimentin as an antibody and then stripped blot using abcam mild stripping method, and probed for actin both times still getting alot of of non specific binding.

The second time i tried running the blot used the same thing for the gel TGS running buffer the same amount of time and transferred it to a PVDF membrane, the difference here was that i used BSA fraction V for the blocking instead of the 5% non-fat milk, I did a 10% BSA solution in PBS-T i left the membrane  in the blocking buffer overnight at room temp using gentle rocking. the next day i washed the membrane 3x for 5 minutes in PBS-t and then proceeded to do the primary incubation with Actin at a 1:500, and 1:1000 concentration in PBS-T with 0.5% milk added for an 1hr:20min with gentle rocking,then i washed the membrane again 3x for 5min, and proceeded to the secondary antibody incubation at a concentration of 1:5000 in PBS-t for an hour again gentle rocking, then i proceeded to do the washing again 3x for 5 min and put the ECL substrate once again and incubated 5 min before i imaged, by the way i am using the bio-rad chemi doc to image.

Hope that was a bit more detailed, i greatly appreciate the help

[bob1]

That description is fine.

Your problem probably lies either with the primary or the secondary dilution.  I would run a sample in multiple lanes on the same gel, transfer, then cut the membrane into strips (one lane per strip) and incubate in different dilutions of the primary (keep the 2ry the same, 1:5000) and see if that makes a difference.  You can then repeat this with secondary starting at 1;5000.  You will probably find that there is an optimal dilution of both.  1:10,000 is a common dilution for 2ries.  Keep the 2ry incubation time to an hour or shorter!.

You can also do more washes either by longer washes or by increaing the number.  4 x 5 min is common.

You can dilute the antibodies in blocking solution, which should help lower the background too.

[Gangwolf]

Sometimes loading too much lysate also causes multiple bands.
I don't know what you did prior to lysing your cells but using trypsing to dislodge the cells could definitely give you multiple bands as trypsin might digest your protein of interest.

[ehernandez4]

Gangwolf, on 21 March 2012 - 06:50 AM, said:

Sometimes loading too much lysate also causes multiple bands.
I don't know what you did prior to lysing your cells but using trypsing to dislodge the cells could definitely give you multiple bands as trypsin might digest your protein of interest.

well i have been using trypsin to dislodge my cells, what would you recommend using instead?

[bob1]

Gangwolf, on 21 March 2012 - 06:50 AM, said:

Sometimes loading too much lysate also causes multiple bands.
I don't know what you did prior to lysing your cells but using trypsing to dislodge the cells could definitely give you multiple bands as trypsin might digest your protein of interest.

This should only happen if you are looking at cell surface markers (vimentin isn't one), don't neutralise your trypsin (by adding FBS containing medium), don't wash the cells before lysing, and don't include protease inhibitors in your lysis buffer.

I routinely use trypsin to lift my cells for western blots and don't have any problems with multiple bands from tryptic digestion.

[Gangwolf]

bob1, on 21 March 2012 - 11:49 AM, said:

Gangwolf, on 21 March 2012 - 06:50 AM, said:

Sometimes loading too much lysate also causes multiple bands.
I don't know what you did prior to lysing your cells but using trypsing to dislodge the cells could definitely give you multiple bands as trypsin might digest your protein of interest.

This should only happen if you are looking at cell surface markers (vimentin isn't one), don't neutralise your trypsin (by adding FBS containing medium), don't wash the cells before lysing, and don't include protease inhibitors in your lysis buffer.

I routinely use trypsin to lift my cells for western blots and don't have any problems with multiple bands from tryptic digestion.

Respectfully, it is not entirely true!
In the past when I worked with a transcription factor and did what you suggested that one should do when using trypsin, the transcription factor was still seen as multiple bands until I stopped using trypsin.
Nowadays, I just lyse the cells directly on the plates/flasks.

[bob1]

Gangwolf, on 22 March 2012 - 02:34 AM, said:

bob1, on 21 March 2012 - 11:49 AM, said:

Gangwolf, on 21 March 2012 - 06:50 AM, said:

I don't know what you did prior to lysing your cells but using trypsing to dislodge the cells could definitely give you multiple bands as trypsin might digest your protein of interest.

I routinely use trypsin to lift my cells for western blots and don't have any problems with multiple bands from tryptic digestion.

Respectfully, it is not entirely true!
In the past when I worked with a transcription factor and did what you suggested that one should do when using trypsin, the transcription factor was still seen as multiple bands until I stopped using trypsin.
Nowadays, I just lyse the cells directly on the plates/flasks.

OK, good to know, just never seen it before - your TFs must be particularly sensitive to trypsin.