Dear all,
I try to detect the LDL receptor in my liver Tumor cells (HEP - G2).
For this. I have two different primary antibodies:
1) http://www.abcam.com/ldl-receptor-antibody-ab30532.html
2) http://www.biovision.com/ldlr-antibody-1296.html
For the SDS - Page (reducing conditions), I use a 8 - 16 % Tris-Glycine-Gel and I also dilute my Protein - sample in an additional reducing Agent.
My total Protein concentration is 10 µg.
Furthermore, I Transfer my Proteins to a PVDF Membrane.
Usually, I dilute my Primary antibody 1:1000 and the second one 1:5000.
After adding the Primary antibody, I incubate the Membrane overnight at 4 °C.
After washing the Membrane with TBS-T, I incubate it with the second antibody for an hour at RT.
My results:
I always get the same bands, no matter which Primary antibody I used.
Curiously, the bands are not the predicted/expected ones (see links above).
I repeated the WB a couple of times and tried to optimize it, but I always get the same (very strong) bands.
Do you have any suggestions?
Best regards
Bastian
Hi Bastian, to me it looks like your secondary is reacting with the heavy and light chains of either endogenous IgG or IgG from the media.
What is your secondary Ab? Has it been immunodepleted against human and bovine IgGs?
HI Bastian
Looks Like you are seeing light chain of 2ry Ab. Do following,
Run a gel and without using primary ab, blot it with 2ry ab, If you still see this band pattern meaning these are all non specific 2ry ab binding.
Try using little less diluted primary ab( 1:500) and more diluted 2ry ab (1:20,000).
I am assuming you are using 3%BSA as blocking agent, Try using some other blocking agent( MILK, Sea BLock) as sometime the Primary ab is not compatible with blocking agent and when you make your primary ab dilution with blocking agent, all your 1ry ab is consumed and you end up with only blocking agent.
Thanks a lot for your answers.
@John:
1) In this case, I tried only one dilution for each AB. I applied biological triplicates for each cell line (HEP G2 and HEK 293) and this is why I have 3 lines for each cell line on the membrane. But I already tried to optimize the Blot and tested several dilutions.
2) The max. dilution for the second AB was 1:5000, maybe I should use a higher dilution.
3) The HEP - G2 cells are my positive controls, because the LDL receptor should be overexpressed in these cells (liver cells). And indeed, you can see a stronger band when using the proteins, which are extractet from these cells (this is baffling to me).
4) I used at least 10 µg, maybe I should use less concentrated proteins in the future
@Steingrimur:
My secondary AB is a goat poly to rabbit hrp - antibody. Before harvesting my cells, I took off the medium. So, there should be no medium ingredients left.
Interestingly, the manufacturer says, that "The antibody may cross-react with immunoglobulins from other species. "
@Mitesh:
Thanks, this seems to be a great idea.
I will try to keep off the primary AB at all. If I don´t get any band, I will try to use the dilutions, which you suggested to me.
If this doesn´t help, I will order a new secondary antibody.
I already use 5 % milk as blocking agent for the membrane. I dilute the antibodies just in TBS-T buffer, without any blocking agent.
I´ll keep you informed.
Best regards
Bastian
Dear all,
I started two WBs this week.
One without the first AB and the other one with a lower dilution of the first AB (1:500) and a higher dilution of the second AB (1:20000).
In this second blot, I varied the protein concentrations as well (low = 5µg, high = 10 µg).
As expacted, I don´t see any band in the first Blot.
In the second Blot, I can see the "usual" and very strong band. But I also see additional bands. One of them is at the top of the blot, at the predictet height.
But what about the other bands, how could I "eliminate" them?
Best regards
Bastian
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