Preparation of Cell Lysate for Western Blot - What did I do wrong? (Nov/12/2007 )
Hello again,
I am still struggling with my Western blot, so please advise me on this.
I keep getting multiple bands (both with lower and higher MW than the protein of interest).
I don’t think it’s because the target in my protein sample has been digested or that the antibody concentration is too high.
Therefore I’ll show you how I prepare the cell lysate, in comparison with the procedure mentioned in Ab datasheet. There are some differences.
(note: cells are grown and incubated with test compounds in 25 cm2 tissue culture flask, and everything is done on ice)
My procedure:
1. Rinse cells with ice-cold PBS
2. Scrape off cells in ice-cold PBS
3. Cebtrifuge 2 minutes 1000 rpm
4. Remove supernatant and resuspend pellet in PBS containing protease and phosphatase inhibitor cocktail tablets
5. Centrifuge again
6. Remove supernatant and resuspend pellet in PBS containing protease and phosphatase inhibitor cocktail tablets
7. Sonicate for 20 seconds
8. Determine the protein concentration
9. Add sample buffer
(Sample buffer: 8% SDS, 40% glycerol, 0.01% bromophenol blue, 0.2 M Tris-HCL pH 6.8, and β-mercaptoethanol 75 µl/ml)
10. Load samples into gel and perform electrophoresis.
Supplier's procedure:
1. Rinse cells with PBS
2. Lyse cells by adding SDS sample buffer (contains 62.5 mM Tris HCl ph 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue)
3. Scrape cells and transfer into microcentrifuge tube. Keep on ice
4. Sonicate for 10-15 seconds
5. Heat to 95-100° for 5 minutes, cool on ice
6. Microcentrifuge 5 minutes
7. Load onto SDS-PAGE gel
The favour I’m asking you: please let me know which step you think is incorrect or might have caused the multiple bands.
The rest of my procedure is exactly the same as the supplier’s. So that’s why I thought the bad results may be due one of the steps above.
Thanks a lot!!!!!
[attachment=3821:MK71029_pAMPK.jpg]
Could your test samples be breaking down your protein?
Does your supplier show a photo of their western?
Is it possible the antibody is not clean?
Has anyone else got a clean western with the same antibody?
This is possible. Do not trust the company's data sheet entirely.
What size is the expected protein? If it is the bigger one, the lower bands could be due to degradation. If it is the smaller one, the upper bands could be due to aggregation. Of course, unspecific antibody is always a possibility. Have you ever tried the method that the data sheet mentioned? Try it and see it it could be of any help. Or else just skip the sonicatior step in your protocol and lyze cells by boiling in sample buffer instead. You don't yet need protein concentration step if you just want to see your antibody works or not, right? Just roughly estimate how much to load or load a series of different amount of sample.
[attachment=3823:Datashee...bbit_mAb.pdf]
Hello, thanks again for all of your help & interest.
I've attached the datasheet of the Ab. It's monoclonal so it should be quite specific.
The protein of interest is 62 kDa. If you have a look at my blot result, it should be somewhere near the big, pink band (the pink band is 63 kDa).
Unfortunately in my lab no one had worked with this Ab, but many literatures use it and manage to get good results.
I've been using this Ab for 1 month. On the first week, I got a nice, single band (see picture below).
[attachment=3824:MK71001_...n_new_Ab.jpg]
(yeah it's not too nice but at least there's only 1 band in each lane)
With time, I got more and more bands (see picture on my first post).
So where does the problem lie: the sample or the Ab?
Thanks!
With time, I got more and more bands (see picture on my first post).
So where does the problem lie: the sample or the Ab?
Thanks!
The picture isn't that clear to me but it looks like you have quite a bit of protein degradation in that blot
Clare
you don't centrifuge to get rid of the cells debris after lysis.
This could be an explaination to the aggregations you see.
centrifuge 12000g 10 min 4°C after lysis.
Hi,
i agree with Missele,
if it is degradation then you should see the smear only starting from the 63kDa band
but the smear is from the start of the blot
so it could be because the cell lysate is not clarified properly or you are overloading the gel with too much cell lysate
try to decrease this and then try again
all the best
Leelaram
aaaah i see i see,,
thanks a lot everyone!
i'm starting to see the light here 
thank goodness for this kind of forum!
one other thing, the supplier boils the sample in the loading buffer, your procedure doesn't. this doesn't just lyse the cells, it also denatures the proteins. you may want to try boiling the sample before loading.