SDS-PAGE (High MW Protein)

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jx_dias
jx_dias's picture
SDS-PAGE (High MW Protein)

I am currently trying to isolate a protein that is above 800kDa, a fairly large protein. I have done the SDS PAGE and membrane transfer a few times. After the membrane transfer, each time I stain the membrane (Ponceau & Coomassie) I get no bands. This meant that transfer time is not sufficient (which is one of the problems I am working on).

But the main problem is that when I've stained the gels (Coomassie) I only sometimes see bands on the gel. When I did a 6% gel, I saw a few bands at the top of the gels, but only in a couple of the lanes. When I did a 4% gel, I didn't see any bands on the gel at all, but there was staining at the bottom of the wells (I also stained the stacking gel this time).

Each time I ran the gel at 200V for 1.5 to 2 hours.

Any help of suggestions would be very much appreciated.

Thanks,

Joshua Dias

pinakin dhandhukia
pinakin dhandhukia's picture
hi! you have very intresting

hi! you have very intresting problem. this type of problem was occured in our lab previously with plant meterial. i think your problrm with the sample prepration..
you does not mention ur extraction procedure and source and concentration of protein u loaded in the gel.. if u provide it to me, may be halpful to have some solution. u can write to me eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%70%69%6e%61%6b%69%6e%2e%64%68%61%6e%64%68%75%6b%69%61%40%67%6d%61%69%6c%2e%63%6f%6d%22%3e%70%69%6e%61%6b%69%6e%2e%64%68%61%6e%64%68%75%6b%69%61%40%67%6d%61%69%6c%2e%63%6f%6d%3c%2f%61%3e%27%29%3b'))...
even you can try with the lowering the voltage or go for gradient of 5 to 10%...

have nice time

pinakin dhandhukia

jx_dias wrote:

I am currently trying to isolate a protein that is above 800kDa, a fairly large protein. I have done the SDS PAGE and membrane transfer a few times. After the membrane transfer, each time I stain the membrane (Ponceau & Coomassie) I get no bands. This meant that transfer time is not sufficient (which is one of the problems I am working on).

But the main problem is that when I've stained the gels (Coomassie) I only sometimes see bands on the gel. When I did a 6% gel, I saw a few bands at the top of the gels, but only in a couple of the lanes. When I did a 4% gel, I didn't see any bands on the gel at all, but there was staining at the bottom of the wells (I also stained the stacking gel this time).

Each time I ran the gel at 200V for 1.5 to 2 hours.

Any help of suggestions would be very much appreciated.

Thanks,

Joshua Dias

Protoplast
Protoplast's picture
This may be totally

This may be totally rediculous to say, but have you in anyway quantified expression of the protein? are you certain it is a stable protein? Is there a test for protein activity or similiar? have you conducted a northern? It could be that the protein is either just not there, or not there in high enough concentrations to visualise on a gel?

jx_dias
jx_dias's picture
We did do a Bradford assay to

We did do a Bradford assay to determine protein concentration of the samples. In each well we loaded 10 micrograms.

Joshua Dias

Protoplast wrote:

This may be totally rediculous to say, but have you in anyway quantified expression of the protein? are you certain it is a stable protein? Is there a test for protein activity or similiar? have you conducted a northern? It could be that the protein is either just not there, or not there in high enough concentrations to visualise on a gel?
ise702
ise702's picture
you can try doing a silver

you can try doing a silver stain which in my opinion is more sensitive than the coomassie stain. The silver stain will allow you to see the bands that you are looking for and for the SDS-PAGE part try using a 10-20% tricine 10 well gel load 30ul of your sample and run at 125 for an hour and a half and from there you can do the silver stain

jx_dias wrote:

We did do a Bradford assay to determine protein concentration of the samples. In each well we loaded 10 micrograms.

Joshua Dias

Protoplast wrote:

This may be totally rediculous to say, but have you in anyway quantified expression of the protein? are you certain it is a stable protein? Is there a test for protein activity or similiar? have you conducted a northern? It could be that the protein is either just not there, or not there in high enough concentrations to visualise on a gel?
saswati1
saswati1's picture
HI, Since 800kd is quite high

HI, Since 800kd is quite high. What marker are you using? If you run too much, the possibility is-rest of the protein which are of much smaller size are already out of the gel. In that case , theoretically you should see some bands at the bottom of the gel. Do you see the transfer of the marker well when you stain ?

pinakin dhandhukia wrote:

hi! you have very intresting problem. this type of problem was occured in our lab previously with plant meterial. i think your problrm with the sample prepration..
you does not mention ur extraction procedure and source and concentration of protein u loaded in the gel.. if u provide it to me, may be halpful to have some solution. u can write to me eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%70%69%6e%61%6b%69%6e%2e%64%68%61%6e%64%68%75%6b%69%61%40%67%6d%61%69%6c%2e%63%6f%6d%22%3e%70%69%6e%61%6b%69%6e%2e%64%68%61%6e%64%68%75%6b%69%61%40%67%6d%61%69%6c%2e%63%6f%6d%3c%2f%61%3e%27%29%3b'))...
even you can try with the lowering the voltage or go for gradient of 5 to 10%...

have nice time

pinakin dhandhukia

jx_dias wrote:

I am currently trying to isolate a protein that is above 800kDa, a fairly large protein. I have done the SDS PAGE and membrane transfer a few times. After the membrane transfer, each time I stain the membrane (Ponceau & Coomassie) I get no bands. This meant that transfer time is not sufficient (which is one of the problems I am working on).

But the main problem is that when I've stained the gels (Coomassie) I only sometimes see bands on the gel. When I did a 6% gel, I saw a few bands at the top of the gels, but only in a couple of the lanes. When I did a 4% gel, I didn't see any bands on the gel at all, but there was staining at the bottom of the wells (I also stained the stacking gel this time).

Each time I ran the gel at 200V for 1.5 to 2 hours.

Any help of suggestions would be very much appreciated.

Thanks,

Joshua Dias

samm
samm's picture
Hi! We often work with 400

Hi! We often work with 400+kDa protein: 4% SDS-PAGE, cold, (either with circulation or in cold room), 100 volts, and we run out all but the largest marker (~230 kDa, prestained). Transfer is done using a wet, rather than semi-dry, set-up, again constant voltage - this time ~30 v overnight. This results in pretty good transfer, but your 800+ protein might be a different proposition!

jx_dias
jx_dias's picture
Well we are using a SeeBlue

Well we are using a SeeBlue Plus2 pre-stained ladder. It goes up to 250kDa and we usually run the marker out of the gel.

I have used the same protein sample to isolate a 25kDa protein and it worked perfectly.

Thanks for the help!

Josh

saswati1 wrote:

HI, Since 800kd is quite high. What marker are you using? If you run too much, the possibility is-rest of the protein which are of much smaller size are already out of the gel. In that case , theoretically you should see some bands at the bottom of the gel. Do you see the transfer of the marker well when you stain ?

pinakin dhandhukia wrote:

hi! you have very intresting problem. this type of problem was occured in our lab previously with plant meterial. i think your problrm with the sample prepration..
you does not mention ur extraction procedure and source and concentration of protein u loaded in the gel.. if u provide it to me, may be halpful to have some solution. u can write to me eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%70%69%6e%61%6b%69%6e%2e%64%68%61%6e%64%68%75%6b%69%61%40%67%6d%61%69%6c%2e%63%6f%6d%22%3e%70%69%6e%61%6b%69%6e%2e%64%68%61%6e%64%68%75%6b%69%61%40%67%6d%61%69%6c%2e%63%6f%6d%3c%2f%61%3e%27%29%3b'))...
even you can try with the lowering the voltage or go for gradient of 5 to 10%...

have nice time

pinakin dhandhukia

jx_dias wrote:

I am currently trying to isolate a protein that is above 800kDa, a fairly large protein. I have done the SDS PAGE and membrane transfer a few times. After the membrane transfer, each time I stain the membrane (Ponceau & Coomassie) I get no bands. This meant that transfer time is not sufficient (which is one of the problems I am working on).

But the main problem is that when I've stained the gels (Coomassie) I only sometimes see bands on the gel. When I did a 6% gel, I saw a few bands at the top of the gels, but only in a couple of the lanes. When I did a 4% gel, I didn't see any bands on the gel at all, but there was staining at the bottom of the wells (I also stained the stacking gel this time).

Each time I ran the gel at 200V for 1.5 to 2 hours.

Any help of suggestions would be very much appreciated.

Thanks,

Joshua Dias

jx_dias
jx_dias's picture
Well I just completed a

Well I just completed a continuous SDS-PAGE gel and the target proteins migrated just into the top of the gel. After that, I tried doing a semi-dry transfer for 5 hours and it didnt work. I am now going to try doing a wet transfer overnight and see what happens. Thank you and everyone else for their help and input.

Joshua Dias

samm wrote:

Hi! We often work with 400+kDa protein: 4% SDS-PAGE, cold, (either with circulation or in cold room), 100 volts, and we run out all but the largest marker (~230 kDa, prestained). Transfer is done using a wet, rather than semi-dry, set-up, again constant voltage - this time ~30 v overnight. This results in pretty good transfer, but your 800+ protein might be a different proposition!
Add colour 2 ur life
Add colour 2 ur life's picture
gel % is ok let me know that

gel % is ok let me know that r u loading the protein sufficient for coomassie staining otherwise try silver ,is ur protein 800kda consist of any multi domain structure ? r u doing native page or sds page ?