Dear all,
I try to prove the presence of the LDL - receptor.
I performed my Western Blot several times, with different antibodies and I always received the same bands.
One of these bands (between 25 and 35 kDa) is very strong and after quite a long research, I found out, that this band is most likely a splice variant of the LDL - receptor.
I would like to verify this assumption by a MALDI - TOF MS analysis. My problem is, that I have no idea, how to extract this specific band from my gele after SDS - PAGE, as there will be many other bands, which will overlap my band of interest. Or is there any fast and easy way to extract the band after incubating the membrane with the AB´s ?
Best regards,
Bastian
Bastian, what does the gel look like when you stain it with silver and/or Coomassie? If you see the same discrete band there, it is worthwhile to perform an in-gel digest with trypsin and analyze with MALDI-TOF using Peptide Mass Fingerprinting and (instrumentation allowing) confirm some of the assigned peptides by MS-MS or at least PSD.
If the band pattern from a global staining looks completely different then your protein of interest is very probably a minority compound and as such not amenable to PMF or MALDI-TOF. Try and find somebody who runs high-quality nanoLC-MS/MS instead which can resolve up to 150 different proteins in a gel band in many cases. Am not sure who would be able to do this in Bielefeld - the Niehaus group might be able to help.
Dear Christof,
thanks for your message.
To be honest, I never stained the gel after performing the SDS - PAGE. But I will do it the next time. So, what if I don´t see the band of interest in such a discrete way as in my Blot?
Could I give it a try to cut out my band and analyse it via MALDI - TOF after digesting it with Trypsin? Through the Trypsin, I should receive a Peptide Mass Fingerprinting, shouldn´t I?
Best regards
Bastian
Bastian, is your antibody good for immunoprecipitation? If so, just IP from a large amount of cells, run 95% of the IP on a gel, stain the gel with something like Coomassie (or silver if you don't have too much material) and then cut out the band and give it for MS. I'd first run a western on the other 5% of the IP just to confirm the band is reactive with your antibody. There will still be other proteins in the region, but your protein should be a majority component during the MS. Remember not to include DTT when eluting after the IP, as you don't want the light chain of IgG which will run around the same size as your protein of interest.
Hi Bastian,
If you already have an antibody against the ~25-35kDa band, you are almost there, to try and answer your question.
Next, you will need to covalently attach the antibody to a support; companies such as Pierce have kits to chemically bind the antibody to a matrix/support. You can also try magnetic beads (Dynal beads from Lifetechnologies) to immobilize the antibody to magnetic beads..If you do this step, as Bryen A Jordan pointed out already, you will be able to prevent light chain IgG (it runs around 25 kDa and smears) from contaminating your band of interest. If you skip this step, you will have IgG contaminating your immunoprecipitation.
Next, you should do an affinity purification/pulldown (already suggested by Bryen above) using your antibody bound on the matrix. It may also be a good idea to carry out the immunopurification using another antibody (non-specific) as control, or if you do not have one, use your specific antibody as IgG alone as control with no protein extract.
Finally, you can visualize the protein of interest and whatever may be specifically and unspecifically bound to your antibody, by staining the gel with a sensitive dye eg., Simply blue (it is about 5 times more sensitive than regular Coomassie Blue stain and is also compatible with downstream mass spectrometry analysis steps). A western blot of a fraction of the purified preparation, should confirm that you have your protein of interest ready for protein identification. This band should be absent in the lane where you have used IgG alone. If you have prestained molecular weight markers, they are useful to use during the comparative Western Blot (membrane bound) vs Stain Protein (on gel) step; you can even run one gel, load most of the preparation on one side, have empty lanes followed by Pre-Stained Molecular Weight Markers, empty lanes and finally, a small fraction of your preparation for Western blot, all on the same gel. After SDS-PAGE, cut the gel in the middle of Pre-stained markers, stain the half of gel that contains most material using protein stain and the other half, use it for transfer onto PVDF. After transfer, proceed with western blot. Having Western Blot side by side with the stained gel can help you align the stained protein and western blot easily.
Once you confirm the position of LDL-receptor, in-gel digestion followed by a quick peptide clean up should result in peptides you can identify with LC-MS/MS or MALDI-TOF/TOF.
Good luck,
Hediye.
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