Anti-Lrp2 / Megalin antibody (ab56014)

Dear,
I'm sorry it took me a while to answer your mail, however these are the answers
to you questions:
1. the other anti-LRP2 which I've been using is from Santa Cruz Sc-25470
2. I was using Passive Lysis buffer for all the samples, except for the human
kidney lysate ab44687, which was loaded on the gel as is.
3. the transfer buffer composition: Tris, Glycine and methanol in dH2O.
4. the transfer was for 2hr in 200mA.
5. the 500-600kDa proteins were transfered OK when I checked with Ponceu red,
however, unfortunately I did not scan the membrane after the dye and continued
to blocking.
6. The membrane was exposed to the film for 1, 5, 15 and 60 min.
7. No primary control was performed.
It is noteworthy that the positive control which was purchased from your company
(human kidney lysate), did not work in the correct band size, and I asked you
before what is the expected size of bands when using this lysate?

ANSWER:

Thank you for providing that extra information.

The band that can be expected with this lysate is the predicted 517 kDa, however, to our knowledge, this lysate has not been specifically probed for Lrp2 and I cannot therefore be sure of this. Additionally, ab56014 has not been used to detect the endogenously expressed protein in western blotting in house (which is also seems to betrue of the Sc-25470). Only recombinantly expressed protein fragments have been detected. This makes performing the western blot considerably easier as the transfer of the smaller recombinant protein is much less problematic than transferring a 517 kDa protein.

Can I ask, are you seeing any bands after performing the western blot or is the membrane completely blank? If you could possibly share an image of the blot obtained that would be very helpful. What was the result of the "no primary" control?

I would advise tryingthe following in order to improve the resultsobserved to date:

1.I would prepare the protein samples by heating for 10 minutes to 70 degrees. If you are not already doing so I would also used a reducing agent in the loading buffer. This is not necessary with the control lysate as this is already included (butthe heating step should still be performed).

2. In order to improve the transfer and binding of the protein to the membrane I would use PVDF membrane and make sure to pretreat in methanol for 1-2 minutes, followed by incubation in ice cold tranfer buffer for 5 minutes.

3. I would use a very low percentage gel in order to improve the transfer, as well as adding 0.1% SDS to the tranfer buffer as this prevents the precipitation of the protein in the gel. I would also remove the methanol from the transfer buffer as this tends to remove the SDS. Methanol is only necessary in the transfer buffer if using a nitrocellulose membrane. Removing the methanol also promotes the swelling of the membrane which allows large proteins to transfer more easily.

I hope these suggestions improve the results observed so far. Performing a western blot with such a large protein will be quite difficult and may require quite a lot of optimisation in order to get a good result.

I look forward to receiving yourreply.

1) Abcam product code ab 44687

2) Abcam order reference number or product batch number
GR 316263
3) Description of the problem: no band / high background (many non-specific
bands) / wrong band size / other?
Wrong band size
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell
lysate
Species : Human kidney cells (HK-2)
Lysis buffer : Passive lysis buffer which contains protease inhibitors
Protease inhibitors:
Phosphatase inhibitors :
Reducing agent :
Boiling for ≥5 min? yes (I have boiled my samples, ab 44687 was loaded as
is without boiling)
Protein loaded ug/lane or cells/lane : 40µg
Positive control : human kidney lysate ab 44687
Negative control : HeLa cells
5) Percentage of gel 5%
Type of membrane nitrocellulose
Protein transfer verified Ponceau
Blocking agent and concentration 5% of non fat dry milk in TBST
Blocking time 1Hr
Blocking temperature room temp.
6) Primary antibody (If more than one was used, describe in “additional notes”)
: rabbit anti human megalin
Concentration or dilution 1:200 , 1:500
Diluent buffer blocking solution
Incubation time overnight
Incubation temperature: 4C
7) Secondary antibody: goat anti rabbit HRP
Species: goat
Reacts against: rabbit
Concentration or dilution 1:25,000
Diluent buffer TBST
Incubation time 1hr
Incubation temperature: room remp.
Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies:
Buffer TBST
Number of washes 4 times
9)Detection method film exposure
10) How many times have you run this staining? once
Do you obtain the same results every time?
What steps have you altered to try and optimize the use of this antibody?
I've used your kidney lysate with two antibodies: the first one was ab56014
(anti human megalin directed at the N-terminal), which I have tried several
times without getting any bands, and the second was another antibody directed
at the C-terminal (not from abcam). The conditions I've used were fairely the
same with both antibodies, however, I did not get any bands with your antibody.
The new antibody gave me wrong bands (˜200kDa instead of ˜600kDa) even when I
used your human kidney lysate.
I would really appreciate your help in this matter.
Best regards,

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to clarify a few details in order to be able to understand the situation more fully and hopefully be able to provide some suggestions in how to improve the results currently observed.

1. could you share with me what the other anti-Lrp2 antibody used was (manufacturers name and catalogue number)?

2. what lysis buffer was used for your other samples?

3. what transfer buffers were used (composition)?

4. how long was the transfer performed for and at what voltage?

5. you mentioned that the transfer was verified by staining of the membrane, do I assume right that the 500-600 kDa region transferred ok? Could you share the images of theseresults with me?

6. how long was the membraneexposed for?

7. has a "no primary" control been performed?

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Dear Tech support

One of our customers has ordered ab56014 anti Lrp2 antibody and Kidney (Human) Tissue Lysate - adult normal tissue.

The customer used the lysate as a controlfor Lrp2 antibody reactivity. The bands received with the lysatewere around 150-200 kda and the customer has expected them to be around 400-600 Kda representing the real size of the protein.

What is the expected band size withab56014 antibody? has the lysate undergone any process that might shorten the protein length?

Thank you for your kind help.

Regards,

ANSWER:

Thank you for contacting us and reporting the problems observed with using anti-Lrp2antibody ab56014 with Kidney (Human) Tissue Lysate (ab44687).

In order to understand the problems your customer is experiencing further could you please pass on the questionnaire attached to this email for them to fill out. Performing western blotting with such a large protein can be very tricky and fully understanding the protocol your customer has used may shed further light on what may be the problem.


I look forward to receiving your reply.