Anti-DCAMKL1 antibody (ab37994)

Thank you for your prompt reply, please find below the conditions used for our experiments, as well as attachments of the western blots and the paper that you requested.



Kindest regards,

1) Abcam product code ab37994 and ab15463



2) Abcam order reference number or product batch number: HGA4500684274



3) Description of the problem: Both antibodies have failed to detect any protein on western blot and immunofluorescence in cell lines fully expected to produce those proteins, as shown in previously published work (please see attached paper Ali et al).



For Western blot:



4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…): Whole cell lysates

Lysis buffer: EBC

Protease inhibitors: Complete protease inhibitor kit.

Phosphatase inhibitors

Reducing agent

Boiling for ≥5 min? yes

Protein loaded ug/lane or cells/lane: 10ul of cell lysate (standardized for protein concentration by Bradford assay.

Positive control: HUH7

Negative control: None



5) Percentage of gel: Protean pre-cast gel 4-20%

Type of membrane: PDVF

Protein transfer verified: yes

Blocking agent and concentration: 10% dried milk powder/TBS-tween

Blocking time: 2 hours

Blocking temperature: room temperature



6) Primary antibody (If more than one was used, describe in “additional notes”) :CK19 and DCAMKL-1

Concentration or dilution: 1:1000 for both antibodies

Diluent buffer: 5% dried milk powder/TBS-Tween

Incubation time: 2 hours

Incubation temperature: Room temperature



7) Secondary antibody:

Species: Goat

Reacts against: Rabbit

Concentration or dilution: 1:2500

Diluent buffer: 5% milk/TBS-tween

Incubation time: 2 hours

Incubation temperature: room temperature

Fluorochrome or enzyme conjugate: HRP



8) Washing after primary and secondary antibodies:

Buffer: TBS-Tween

Number of washes: 2x quick washes, followed by 1x 10 minute wash, then 3x 5minute washes



9)Detection method: ECL



10) How many times have you run this staining? Once for Western blot and once for immunofluorescence

Do you obtain the same results every time? -

What steps have you altered to try and optimize the use of this antibody? –



Attached document shows these results, as well as confirmation of even loading (GAPDH).



For Immunofluorescence



4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…): Cells grown on cover slip in 6-well plate, then treated with 4% paraformaldehyde, prior to membrane disruption with 0.2% TRITON-X100 for 5 minutes.

Positive control: HUH7

Negative control: None



5) Blocking agent and concentration: Blocking carried out with 10%FCS/PBS alongside primary and secondary antibody probing as below.



6) Primary antibody (If more than one was used, describe in “additional notes”) :CK19 and DCAMKL-1

Concentration or dilution: 1:200 (CK19) and 1:250 (DCAMKL-1).

Diluent buffer: 10%FCS/PBS

Incubation time: 1 hour

Incubation temperature: Room temperature



7) Secondary antibody:

Species: Goat

Reacts against: Rabbit

Concentration or dilution: 1:200

Diluent buffer: 10%FCS/PBS

Incubation time: 1 hour

Incubation temperature: room temperature

Fluorochrome or enzyme conjugate: ALEXA-FLUR 594



8) Washing after primary and secondary antibodies:

Buffer: PBS

Number of washes: 3



9)Detection method: Fluorescent light microscope

10) How many times have you run this staining? Once for Western blot and once for immunofluorescence

Do you obtain the same results every time? -

What steps have you altered to try and optimize the use of this antibody? -

ANSWER:

Thank you for taking the time to complete our questionnaire and sending me the publication. I am sorry to hear you have had difficulty obtaining satisfactory results from these antibodies.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.


Although the other antibody has worked well using this procedure, individual antibodies will often require optimization. I would like to offer some suggestions to help optimise the results from ab15463 and ab37994.

1.) I suggest to use a different positive control than HUH7 cells. As shown in figure 1 of the publication provided HUH7 cells do not show any expression of CK-19 and a only a very low expression of DCAMKL-1. I can recommend to use HepG2 cells or shin for ab15463 and MCF for ab37994 as indicated on the respective datasheets.

2.) I also can recommend to incubate primary antibodies over night at 4C. This will ensure the best saturation and specificity of the antibody.

3.) For the WB, I suggest to use less than 10% milk powder as blocking agent (maybe try a different blocking agent like BSA which can sometimes have a big impact on signal strength).

4.) For ICC/IF I can recommend to try a milder permeabilisation agent./ For example 0.1 Tween in the blocking buffer for 1 hour.


I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again.

What is the immunogen sequence?

ANSWER:

Thank you for contacting us.

We know that the antibody recognizes the 2 isoforms: AL, BL (which corresponds to isoforms 2 and 4) due to sequence homology.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.