Silver stain Protein detection - Silver stain problem (Mar/23/2007 )

dear all

I have a recurrent problem with my silver staining of my proteins... i have also attached a picture of the staining. you see there is a background in half of the gel and the lower half is lighter. and everytime i have this problem the proteins in the lower half is not stained properly. i have checked a lot of parameters and then i also tried alternative staining (Deep purple staining kit) and then i saw that i have the same "double-phase" problem even in a different technique. now i am have no idea what this problem is... has anyone encountered this problem? does anyone have suggestions about what it can be and how it can be solved....

thank you for your suggestions

regards
Raj

---

this is a problem caused by one or more of the buffers used with your gel. first check the appearance of your buffers (growth, floaters, color). then check the pH of your buffers.

if you pour your own gels, did you add sds to the gel solutions?

we've had this happen on occasion and it usually caused by one of the buffers.

---

thank you for your ideas... but i use NuPAGE precast bis-tris gel from invitrogen. it is run in NuPAGE MOPS SDS running buffer. what is the pH you suggest for the running buffer wich could circumvent the problem?

and you say buffers (in plural). i dont use any buffer other than the running buffer after which the gel is subjected to silver staining... could you please clarify.

DTT or beta-Mercaptoethanol? which one do you suggest is better? could that be the cause of this problem?

thank you for your prompt response...

regards
rajgene

---

electrode buffer pH depends on the buffer system being used. for sds it is usually 7.9-8.7. laemmli is ~8.3.

there can be several buffers used in a gel. running (or separating) gel buffer, stacking gel buffer, sample buffer and electrode buffer. the system i use most has two different electrode buffers for a total of five different buffers (and pHs).

how old are your gels and buffer?

what is the quality of water you use to dilute your electrode (reservoir) buffer?

is the water quality variable?

we use bme but dtt may actually be better (and more expensive). i don't think that it is your problem because it wouldn't be as smooth across the gel if it were (because of the sample wells). although reducing agents cause their own problems, you can get bands in the 60-68 kDa range showing up because of dust dissolved in the agent.

all in all, it still looks like a buffer problem to me.

---

hi

thanks a lot for these suggestions. i will check these. i use milliQ water and the precast gels have its own runing buffer(20x) and sample buffer. i make fresh working solutions and run my gels...

i will verify the parameters you suggested and let you know the result...

Thank you for toubleshooting

Regards
Raj

---