This might indicate to a severe degradation of your protein producing an immunologically positive 30K fragment. Try to use protease inhibitor cocktail at the stage of extraction / lysis of your cells. This might help.

I agree that you should always use protease inhibitors when you are processing your cells or tissues.  I would not be too concerned with your blot having extra bands.  It could certainly be protein degradation and or different forms of your protein.  You did the right thing in running only your secondary antibody to look for unspecific bands.  Since you have your bands of interest present at 130 kDa and in between 55 and 70 kDa, I would not worry about the band at 30 kDa.  If you had more of a ladder effect of bands surrounding your bands at those molecular weights of interest, there could be problems.

Viktor is right, you should try protease inhibitors during the preparation of the LDLR.

Interestingly this band doesn´t increase when you stimulate the HepG2 cells. Do you prepare membrane proteins or whole cell extracts?

Thanks for your answers.

I already added a Protease Inhibtor Cocktail to my Lysis buffer.

As you suggested, it seems that the additional band at 30 kDa is a splice variant of the LDL receptor, which is also Protein coding.

The Ensembl results would perfectly underpin this thesis:

http://www.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000130164;r=19:11089362-11133816

The splice variant, whose Protein contains 309 aa, has got a approx. weight of 34 kDa.

@Michael: I prepared whole cell extracts. Would you recommend a higher Protein concentration?

Best regards

I wouldn´t increase the protein concentration. You can detect the receptor and with a higher concentration you will only get more background.

What is your nitrocellulose-blocking solution? If you use BSA try with defatted powdered milk at 3%.

You may also try with ligand blotting if radiolabelled LDL are available.

Good luck!

Thanks for your answers!

@Michael: I think so, too. Would you be satisfied with the additional bands or would you go on to optimize the blot?

@Giuseppe: I use 5 % of milk powder for the blocking step. I dilute the AB only with TBS-T buffer, without any blocking solution.

Do you think, that I should add blocking solution to the dilution as well?

I agree with victor, it should be a result of severe degradation of your protein. it is very prominent so i don't think that it can be some thing not specific, also its intensity decrease in the Hela low just like your protein so it mostly related to it. 

Sometimes the antibody used for detection has some non-specific interactions. Try saving your primary antibody solution with Sodium Azide for keeping it fresh and reuse it. As you re-use it the background will be less and less.

@ Dianna What concentration of sodium azide is good for saving primary antibody solution?

Use 0.01% w/v Sodium Azide for your primary antibody and store at 4C.

What blocking buffer are you using? Blocker can have a HUGE, unexpected effect on Ab specificity. In a different blocker, you might get a much cleaner blot. We have seen this many times. In our lab, BSA is usually the worst blocker and gives the most background banding. The Biovision datasheet for your Ab doesn't say what blocker they used to generate the sample data - but you could call Biovision tech support and ask.

These papers show the dramatic difference that blocking buffer choice can have on Ab specificity: 

http://www.ncbi.nlm.nih.gov/pubmed/18563731

http://www.springer.com/cda/content/document/cda_downloaddocument/9781617792854-c1.pdf?SGWID=0-0-45-1185840-p174124972

Hi Bastian,

Are you running reduced samples?  From my reading, the LDL-R contains many cysteine residues which may be the reason you are seeing unspecified bands due to breaking of disulfide bonds.  I would try running your samples under non-reducing conditions, ie, without b-mercaptoethanol or DTT.   Abcam also reported similar bands with one of their LDL-R antibodies:  http://www.abcam.com/ldl-receptor-antibody-ep1553y-ab52818.html#description_images_2

Hope that solves the problem.

First of all, thanks for your answers!

@Dianna: I already use Sodium Azide for the storage.

@Amy: For the blocking step, I dilute milk powder (5%) in TBS - T. Do you dilute the AB´s only in TBS-T or do you add milk powder as well? If so, which percentage do you use? Thanks for the papers!

@TIna: Yes, I use a reducing agent and a reducing gele as well. The funny thing is, that I already used the AB from Abcam and received the same bands as for the AB from Biovision.

I found someone, who will check the strong band via MALDI - TOF MS. Does anyone of you have experience with the analysis of WB bands via MALDI - TOF?

Best regards

Bastian

Bastain,

It's not surprising that you saw the same bands with abs from different companies.  If you are running reduced samples you will likely see the same pattern.  Before trying MALDI-TOF, I would re-run samples that have been prepared under non-reducing conditions.  That would certainly be much easier. 

Also, I dilute my antibodies in TBS-T with either 5% milk or 5% BSA, depending on the protocol for that antibody.  I always dilute my secondary antibodies in TBS-T with 5% milk powder.