Is it possible this protein has been post translation modified in the mouse tissue?
or the antibody works in cell culture system but not good in the mouse tissue?
or something else?
Edited by ucsd, 23 July 2009 - 09:03 AM.
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4 replies to this topic
#1
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Posted 23 July 2009 - 08:53 AM
I have done a western blot for a known protein and found in the mouse cell culture system, the protein band is correct and it is around 65KDa. However when I do the same protein using the same antibody but using mouse tissue (liver ,muscle and fat), I found the protein became around 75KDa. Why the same protein using the same antibody have the difference between mouse cell culture and mouse tissue.
Is it possible this protein has been post translation modified in the mouse tissue?
or the antibody works in cell culture system but not good in the mouse tissue?
or something else?
Is it possible this protein has been post translation modified in the mouse tissue?
or the antibody works in cell culture system but not good in the mouse tissue?
or something else?
#2
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Posted 24 July 2009 - 03:06 PM
Few ideas.
-First, the protein quantity in the tissue will be drastically higher than in the cell culture. Higher protein contents tend to run differently than low protein content. 10kDa seems a bit much of a difference, however. Perhaps try reducing the amount loaded for the tissue sample and see how it runs?
-Second, you may have different lysate conditions. For instance, varying amounts of salt or detergent will make the bands run to slightly different weights. Do you use the same lysing procedure for both tissue and cell culture?
-Are you using a stained/dyed ladder? The dyes used to color ladders affect how they run in a gel, which is why the weights are listed as "apparent weights" rather than true molecular weights. The "apparent weights" can be variable depending on the exact makeup of your acrylamide gel (acrylamide percentage, pH, glycine vs. tricine, etc)
-Post-translational modifications are not likely in this scenario. 10 kDa seems way too large for a post-translational modification.
-Perhaps you have a unique isoform in the mouse tissue not seen in your cell line? Is your cell line the same host (ie. both mice?)? If so, this seems like an unlikely possibility, at least without also seeing the 65 kDa band.
-Did you run these on the same blot or different blots? It may just have been differences in the gel composition.
-First, the protein quantity in the tissue will be drastically higher than in the cell culture. Higher protein contents tend to run differently than low protein content. 10kDa seems a bit much of a difference, however. Perhaps try reducing the amount loaded for the tissue sample and see how it runs?
-Second, you may have different lysate conditions. For instance, varying amounts of salt or detergent will make the bands run to slightly different weights. Do you use the same lysing procedure for both tissue and cell culture?
-Are you using a stained/dyed ladder? The dyes used to color ladders affect how they run in a gel, which is why the weights are listed as "apparent weights" rather than true molecular weights. The "apparent weights" can be variable depending on the exact makeup of your acrylamide gel (acrylamide percentage, pH, glycine vs. tricine, etc)
-Post-translational modifications are not likely in this scenario. 10 kDa seems way too large for a post-translational modification.
-Perhaps you have a unique isoform in the mouse tissue not seen in your cell line? Is your cell line the same host (ie. both mice?)? If so, this seems like an unlikely possibility, at least without also seeing the 65 kDa band.
-Did you run these on the same blot or different blots? It may just have been differences in the gel composition.
#3
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Posted 24 July 2009 - 04:16 PM
Firstly Thanks very much for your ideas.
I use 50 ug liver tissue, 30 ug muscle tissue and 20 ug fat tissue for western blot. I use around 10 ug or 15 ug cell lysate for western blot. Maybe you means that this protein is more accmulated in tissue.
Tissue come from mice and cells is mouse 3T3-L1 cells. so they are same species. You are right, I use different lysis buffer for tissue and cell.
I did not run them in the same gel or blot, but the gels I used were bought from the company and the same catlog number. I think both gel should be the same and the prestained marker should show the same pattern of ladders
I think I should try both in the same gel and at the same time decreasing the amount of tissue.
Do you think I need try another company's antibody?
I use 50 ug liver tissue, 30 ug muscle tissue and 20 ug fat tissue for western blot. I use around 10 ug or 15 ug cell lysate for western blot. Maybe you means that this protein is more accmulated in tissue.
Tissue come from mice and cells is mouse 3T3-L1 cells. so they are same species. You are right, I use different lysis buffer for tissue and cell.
I did not run them in the same gel or blot, but the gels I used were bought from the company and the same catlog number. I think both gel should be the same and the prestained marker should show the same pattern of ladders
I think I should try both in the same gel and at the same time decreasing the amount of tissue.
Do you think I need try another company's antibody?
polyfractal, on Jul 24 2009, 04:06 PM, said:
Few ideas.
-First, the protein quantity in the tissue will be drastically higher than in the cell culture. Higher protein contents tend to run differently than low protein content. 10kDa seems a bit much of a difference, however. Perhaps try reducing the amount loaded for the tissue sample and see how it runs?
-Second, you may have different lysate conditions. For instance, varying amounts of salt or detergent will make the bands run to slightly different weights. Do you use the same lysing procedure for both tissue and cell culture?
-Are you using a stained/dyed ladder? The dyes used to color ladders affect how they run in a gel, which is why the weights are listed as "apparent weights" rather than true molecular weights. The "apparent weights" can be variable depending on the exact makeup of your acrylamide gel (acrylamide percentage, pH, glycine vs. tricine, etc)
-Post-translational modifications are not likely in this scenario. 10 kDa seems way too large for a post-translational modification.
-Perhaps you have a unique isoform in the mouse tissue not seen in your cell line? Is your cell line the same host (ie. both mice?)? If so, this seems like an unlikely possibility, at least without also seeing the 65 kDa band.
-Did you run these on the same blot or different blots? It may just have been differences in the gel composition.
-First, the protein quantity in the tissue will be drastically higher than in the cell culture. Higher protein contents tend to run differently than low protein content. 10kDa seems a bit much of a difference, however. Perhaps try reducing the amount loaded for the tissue sample and see how it runs?
-Second, you may have different lysate conditions. For instance, varying amounts of salt or detergent will make the bands run to slightly different weights. Do you use the same lysing procedure for both tissue and cell culture?
-Are you using a stained/dyed ladder? The dyes used to color ladders affect how they run in a gel, which is why the weights are listed as "apparent weights" rather than true molecular weights. The "apparent weights" can be variable depending on the exact makeup of your acrylamide gel (acrylamide percentage, pH, glycine vs. tricine, etc)
-Post-translational modifications are not likely in this scenario. 10 kDa seems way too large for a post-translational modification.
-Perhaps you have a unique isoform in the mouse tissue not seen in your cell line? Is your cell line the same host (ie. both mice?)? If so, this seems like an unlikely possibility, at least without also seeing the 65 kDa band.
-Did you run these on the same blot or different blots? It may just have been differences in the gel composition.
#4
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Posted 25 July 2009 - 11:20 AM
I'd hold off on purchasing another antibody for now, as I don't believe that is the problem. Based on what you said, it is most likely a difference in protein quantity and lysis buffer. For instance, if you were to nanodrop the tissue lysate vs. the cell culture lysate, you would find that the protein mg/mL is much higher in the tissue lysate, which is likely contributing to the different band weight.
I agree with your plan. Try running both on the same gel and reducing the tissue sample.
Also note, your tissue band will likely be very intense while your cell culture band very light, since they are being imaged on the same gel. The increased protein content of the tissue will correlate to an increase in intensity, thus scaling down the less intense cell culture. Not a problem, just something to be aware of. If you are quantifying, loading controls should be used.
I agree with your plan. Try running both on the same gel and reducing the tissue sample.
Also note, your tissue band will likely be very intense while your cell culture band very light, since they are being imaged on the same gel. The increased protein content of the tissue will correlate to an increase in intensity, thus scaling down the less intense cell culture. Not a problem, just something to be aware of. If you are quantifying, loading controls should be used.
#5
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Posted 23 October 2009 - 05:40 PM
Hi USCD,
May I know how you extract protein from adipose tissue for western blot? I have a lot of trouble trying to get good protein from adipose tissue. Do you do a collagenase disgestion? Could you describe how you get protein for WB after you excise fat pad from mouse? The more details the better. Thank you very much!
May I know how you extract protein from adipose tissue for western blot? I have a lot of trouble trying to get good protein from adipose tissue. Do you do a collagenase disgestion? Could you describe how you get protein for WB after you excise fat pad from mouse? The more details the better. Thank you very much!
