Cant see my molecular weight markers on PVDF

[shikimate]

I have been trying to transfer minigels after running SDS-PAGE this week and I keep having the problem of not seeing my MW bands on the PVDF. I am using two kinds of MW markers, one is from bio-rad, and another is the ultra low molecular weight range marker from sigma aldrich. For both markers, I cannot see the bands on the membrane, and they do not appear on the whatman paper behind my membrane either. I am also hesitant to probe my membrane with primary antibody because it is really hard to obtain, and it would be a waste of solution if there was no target on my membrane I think.

just to give you an overview of what I did; I ran samples containing a 1 kDa protein and my markers on the minigel, and then I transferred overnight (16 hours) at 30 volts using transfer buffer containing methanol. I also wet my membrane for about a minute in 100 % methanol before running, and I am pretty sure my electrodes and sandwich were set up correctly. So is there anything else that could be going wrong here ?

Lastly, I read another post on here where someone suggested using ponceau red to stain the membrane and check if the proteins were on the membrane. If I only have coomasie brilliant blue available, could I use that and successfully destain my membrane ?

[relaxin]

Are your Markers pre-stained? Your peptide is only 1 kDa, it may run out of the gel or may not bind to PVDF membrane. Coomassie blue stain will not work. You need reversible MemCode™ Stain from Pierce.

You may try to probe with antibody against a reference protein (beta-actin) first. If it works, then probe with your precious antibody.

[shikimate]

Are your Markers pre-stained? Your peptide is only 1 kDa, it may run out of the gel or may not bind to PVDF membrane. Coomassie blue stain will not work. You need reversible MemCode™ Stain from Pierce.

You may try to probe with antibody against a reference protein (beta-actin) first. If it works, then probe with your precious antibody.

Hi Relaxin,

Thank you for your reply. Actually, I have found papers showing that western blotting can be used to detect microcystin-LR which is 0.995 kDa using nitrocellulose membrane. I did try to contact the authors of one particular paper to ask what conditions they used but I have not heard back I also stained some of my mini-gels using coomassie blue, and I saw diffuse bands in the approximate 1 KDa location I was expecting for microcystin (the fact that the bands were diffuse leads me to believe that it is microcystin as we think there are multiple congeners in my samples with slightly different molecular weights) , so I believe that the microcystin may not necessarily run out of the gel. In addition, I usually stop running my gels when the dye front is 2/3 down the length of the gel to reduce the risk of that happening.

As for my markers, yes they are prestained. I believe the ultra-low molecular weight range marker is not "compatible" with regards to western blotting according to sigma aldrich people, but I expected the bio-rad one to work.

[relaxin]

If you have prestained markers, then you will be sure that the 1 kDa peptide did not run out of the gel. If after transfer, the markers are not visible either on the membrane or gel, then they may simply pass through the membrane.

If you repeat the experiment, try put two pieces of membrane and shorter time of transfer (less than 1 hour at 200 mA).

[mdfenko]

on top of relaxin's recommendation of a shorter transfer time...

what is the pore size of your membrane? for 1 kDa protein i would not use a membrane with a pore larger than 0.2um.

methanol in the buffer is not necessary if you do not add sds to the buffer (used to facilitate transfer of large proteins), but should not hurt.

for small proteins and peptides you may want to try tris-tricine sds-page. it is far superior for the separation of low molecular weight proteins and may give you sharper bands.

[shikimate]

Thanks guys, I am going to try and repeat the experiment again in that case with both of your suggestions. The pore size of the membrane I am using is actually 0.2 um. Is this sufficient given that the peptide i am working with is 7 amino acids long ?

One more thing I wanted to note though; the lowest molecular weight band on my prestained marker is 1.1 KDa , and I did observe this one on my mini-gel after SDS-PAGE. Is this the one I am supposed to look for to ensure the peptide didnt run out of the gel ?

[relaxin]

Yes, always look for the marker whose size is closed to the peptide of interest. If it does not run off the gel, the peptide of interest may still be on the gel.

If you can synthesize a peptide of 7 residues and label it with biotin, you can monitor the transfer process and its ability to bind the membrane with streptavidin-conjugated HRP.

[mdfenko]

i used to separate enkephalins with tris-tricine gels.

0.2um pore is okay. you can get smaller pore but that shouldn't be necessary. i used semi-dry transfer for 15-20 minutes at the recommended current density (mA/cm2) for the apparatus.

[shikimate]

i used to separate enkephalins with tris-tricine gels.

0.2um pore is okay. you can get smaller pore but that shouldn't be necessary. i used semi-dry transfer for 15-20 minutes at the recommended current density (mA/cm2) for the apparatus.

Yes, a 0.2 um pore size with a 15 minute transfer time did work. I saw bands corresponding to the microcystin in all of my samples when I probed with antibody. I think transfer time was the main culprit, thanks for the tip !

[kavizz]

Can you please share your protocol in detail. I am trying to detect a 7kDa protein and am having problems with it.

Thanks!

[mdfenko]

Can you please share your protocol in detail. I am trying to detect a 7kDa protein and am having problems with it.

Thanks!

tell us what you are doing and we can make suggestions

[kavizz]

I am trying to detect a 7 kD FLAG tagged protein by western. I know that protein is expressed (by IF).
Gel: 10-20% tris-tricine
Membrane: Nitrocellulose 0.2 micron
Transfer: wet transfer with tris/glycine/methanol for 45 minutes at 100V
Prestained markers as low as 5 kD are transferred well and Ponceau staining looks good.
Western works well but cannot see this 7 kD band at all.

Should I change the transfer time?

[mdfenko]

you mention that ponceau staining looks good, does that mean you see your 7kDa protein with ponceau?

if so, are you sure your antibody can be used for western blotting? it may be good for native but not for denatured protein.

[kavizz]

No, I don't see my 7 kD protein by Ponceau.

Western is using an antibody to the FLAG tag and it works great.

[mdfenko]

are you seeing low mw standards on the membrane (lower than 7 kDa)?

you can try using a second membrane to back up the first to determine if you are blowing your sample through.

also, stain the gel after transfer to ensure that your protein of interest has exited the gel.

these two checks will help you to determine if you need to adjust transfer time.