p21, p27, Phos-Akt & Phos-Erk 1/2 Expression in KASUMI 1 Cells - p21, p27, Phos-Akt & Phos-Erk 1/2 Expression in KASUMI 1 Cells (Nov/29/2010 )
I am working on the Acute Myeloid Leukemia (AML1-ETO) cell line KASUMI 1. I have a lot of problems with p21, p27, phos-Akt and phos-Erk 1/2 detection by Western Blotting in this cell line, both in untreated & treated cells. I treat cells with drugs in a time dependent manner. I have changed, and tried all possible variants in the blotting protocol starting from Protein extraction to developing.
The reagents that I use are:
1. Electrophoresis buffer: TGS (Fluka - 51306).
2. Transfer buffer: TG (Bio-Rad 161-0771)
1. Primary Antibodies - p21 (CS#2947); p27 (CS#2552); phos-Akt (CS#4060) & phos-Erk 1/2 (CS#9101) tried diluting both 1:1000 & 1:500 in 5% milk.
2. Secondary Antibody (NA934V) tried diluting 1:10,000, 1:5,000 & 1:2,500 in 5% milk.
3. ECL (sc#2048).
4. Nitrocellulose membrane (Bio-Rad: 162-0115).
Do anyone of you have any suggestions...comments...idea/s as to how to tackle with the detection problem of the above 4 molecules - p21, p27, phos-Akt & phos-Erk 1/2 in KASUMI 1 cells???
Thanks in advance!
what are the sizes of the proteins you are transferring? sds is usually only added to the transfer buffer to assist transfer of large proteins and not more than 0.05% or it may interfere with protein binding.
have you stained the membrane with ponceau s after transfer to confirm?
have you stained the gel to make sure that the proteins exited?
what is the pore size of the membrane? if your proteins are small then you may want to use 0.22um or smaller.
Thank you for the reply. The molecular weights are: p21=21 kDa; p27=27 kDa; Akt=60 kDa; Erk 1/2=44/42 kDa respectively. Yes, I always stain the membrane with Ponceau S to confirm the transfer efficiency, and the gel with Coomossie brilliant blue to confirm the exit. The pore size is of 0.45uM (Nitrocellulose). So you think these might be some of the hindering factors??
if you have confirmed transfer of the protein and get no immunostaining then either your primary antibody or secondary antibody is not working.
are the primaries suitable for western blot (check the data sheet that comes with them)? keep in mind that sds-page denatures the protein.
you can test for this with a dot blot. apply your proteins, native and denatured, to a membrane and process as you would a western.
does the secondary antibody work properly with other antibodies? if not then either the antibody itself or the conjugated enzyme has gone bad.
if transfer is poor then you may want to try a smaller pore membrane.
I always add a positive control with my samples. So when I develop the membrane, I get to see the band in the positive control lane!! (of course sometimes I don't get see the band in the positive control also...shockingly!!!). So don't you think there might not be problem/s with any of those you have mentioned - Primary Abs, Secondary Abs, ECL/conjugated enzyme (I have checked the datasheets of all antibodies, and other reagents, and they are suitable for Western blotting)??
if you detect the protein in the positive control but not in the sample then you have too little of the protein present to detect.
if you don't detect the positive control then there is something wrong with one of the antibodies or the detection method (reagent, timing,...).
I think it might be the first case! I think the phosphorylation level could be too less!! ... Can you enlighten me more on Dot-blot?!
Thanks, and have a great weekend
for a dot blot, you spot a membrane with equal volume of each condition and let it adsorb (blotting paper under the membrane will draw the liquid through the pores, the protein should bind).
as i said before, you should use samples treated with and without loading buffer (keep the protein concentration the same, dilute the native as the denatured is diluted).
then process the membrane the same way that you would a western blot.
Ok. Will try these, and let me see. Lets discuss once I get the results. Thanks!
Hi! Am back ... I did the Dot blot with the Native protein samples. After developing, I could see only the circumference of the blot being developed (like a hallow circle), and not the usual way "a dark Blot"