Troubleshooting SDS-PAGE of total protein extraction from sea anemone Aiptasia?

I am new to SDS-PAGE and hope someone can help with a persistent problem I am experiencing.

The dye front is stopping about 3/4 of the way down the gel and doesn't seem to move further down than this. Looking at the gel there appears to be a 'ridge' where the dye front is stopping although it doesn't feel like a physical ridge (see photos attached).

I've tried using fresh plates, buffer and protein samples and used a different power pack but this hasn't helped.

Thanks in advance for any advice

Dear,

Whenever I run an SDS-PAGE, I get an unwanted vertical line appearing at the bottom of the gel. Does anyone have any idea why this happens?

Any comments or suggestions would be highly appreciated.

Thanks,

I would like to check protein profiling of plant protein through SDS PAGE but whenever i run SDS got two unwanted lines within the gel which hampered protein running on gel.. please anyone give me suggestion to resolve this...

for your observation a picture of the gel is attached here with.. first line is of marker...

thanks

I did acetone precipitation and re-suspended my pellet into PBS. I then boiled my samples at 95 degrees for 5mins but my gel have smears all down. I have ran my gel at 200V.

Which is a better option to run SDS PAGE gel...constant current or constant voltage.

Please mention the values and give a reference if possible.

After immunoprecipitation with specific antibody and Protein G, samples were eluted with elution buffer, SDS sample buffer, and reducing agent at 70 °C for 5 minutes. Also, samples were incubated at 95 °C for 10 minutes before loading on SDS-PAGE.

The bubbles did not exist in the gel, and the replicate experiment shows the same result.

This is confusing because the left lane is a negative control, and the right lane is a positive control that should show immunoprecipitated protein.

Is it an protein aggregation?

Hi!

I'm new to making my own gels, so I can't fully eliminate the possibility that the gels I am making are the problem. But, I keep getting this issue with my gels where I am unable to see clear bands because of what I can only guess is a type of smearing. I've changed the APS and TEMED amounts. I've prepared new solutions for making the gels. I've tried different ways of mixing. I've prepared new running buffer. I typically run starting with 100 V and then increase up to about 120-130 V after 30 minutes. At this point, I'm thinking it has something to do with my samples but I don't really know so I am open to hearing your ideas and thoughts. I've attached examples of the gels i've been getting.

Hello,

I'm having trouble with SDS PAGE gel, as you can see some band is formed at the bottom and the proteins can't go through it so they are getting compressed .

I replaced every ingredient of the gel but I'm still getting this band (recipe is in the picture).

Running buffer is composed of : 3.0 g of Tris base, 14.4 g of glycine, and 1.0 g of SDS in 1000 ml of H2O. pH= 8.3 .

250 mM Tris–HCl (pH 6.8)

8% (w/v) sodium dodecyl sulfate (SDS)

0.2% (w/v) bromophenol blue

40% (v/v) glycerol

20% (v/v) β-mercaptoethanol

What can be the problem?

Hi! Just want to see if anyone has some experience how protein degradation looks like in the gel. Attached is a 18% SDS-PAGE for my Ni-NTA purification process for a ~9 kD protein expressed in BL21. The target band looks smeared. I am sure it's not sample overloading (~ 1 microgram loaded), so I am suspecting it is some degradation? (I added PMSF and benzamidinium chloride before lysis) Or is it possible to be some gel running technique issue (but all other bands looks fine)? Thanks a lot!