Recently, I run SDS-PAGE (Protiens extracted from NB4 Cell). proteins do not run in SDS-PAGE I think that there was a problem in extraction of proteins phase. Could you help me with this problem?
Is this a Ponceau staining?
If yes, then your proteins are present, the red band, but did not separate on the gel, and apparently migrated very slowly.
Check the pH of the solutions you have used to prepare the gel. Make sure you have the correct amount of SDS, in the gel and sample buffer.
I don't think this migration problem comes from the lysate preparation, but rather form the way you prepared your sample before loading.
HI,
What extraction procedure do you use? Cells can be easily lysed with many simple buffers. We use buffer with following composition 20nM tris pH7.4, 150mM NaCl, 1nM EDTA, 2mM DTT and 5.5% triton x 100. The cells are homogenized, centrifuged and used. The centrifugation is crucial as it removes insoluble components, nucleus and so on. Sometimes it will be helpful to mildly sonicate the extract. The problem here is likely due to the post sample preparation i.e after SDS sample buffer is added. Centrifugate the sample after heating and then load and check.
Is this a Ponceau staining?
If yes, then your proteins are present, the red band, but did not separate on the gel, and apparently migrated very slowly.
Check the pH of the solutions you have used to prepare the gel. Make sure you have the correct amount of SDS, in the gel and sample buffer.
I don't think this migration problem comes from the lysate preparation, but rather form the way you prepared your sample before loading.
Hello. thanks for your help.
@ Cyrille Girard : Yes, the membrane staining with Panceau S. PH of solution is ok. but there was long time between extracted protein (keep in -80) and sample buffer addition to sample (before the SDS-PAGE running).
thanks all. I have to re-make solutions and buffers.
Hi all recommendation are valid, I am agree with @cyrille girard, probably the solutions you are used, but also check the electrophoretic camera, sometimes if there is some problem in the electronic system, the migration are affected badly. On the other hand check the amount of glycerol of your buffer sample, could be possible that part of your sample are coming out of the well (in case you are working with a semi-purified sample) and you are seeing an specific protein at that molecular weight
Meysam, I guess you forgot to add SDS in your gel, but added it to you samples. So the charge was enough to pass the distance you observed
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