Anti-Calreticulin antibody - ER Marker (ab4)

, I'm trying to measure human estradiol in salivary samples using 3H RIA.  I ordered an antibody from MP Biomedicals, only to discover that it was too dilute for my assay.  Is there any chance I could purchase a small volume of this antibody to make sure it will run in my system? Thanks in advance!

ANSWER:

Thank you for contacting Abcam. We do not stock any sample sizes of our antibodies, but we do cover our antibodies under our Abpromise. In this case ab116648, anti-Estradiol antibody, is guaranteed to work in RIA applications. ab116648 has a total of 100ug at a concentration of 1mg/ml, hopefully this would be at a high enough concentration for your needs. If there is anything else I can help you with, please let me know.

I have incubated the cells with the primary antibody for 1 hour at 4 degrees celsius(I have used this protocol also with other AbCam antibody). I have used the secondary antibody other times with other primary antibody and it works properly. If you need further informations, please do not hesitate to contact me.

ANSWER:

Thank you for your e-mail and for replying to my further questions.

Temperature and incubation period are two crucial factors in the staining procedure. If you incubate the cells with the primary antibody at lower temperature i.e. at 4oC, it is important to perform the experiment overnight for at least for 12-18 hrs, 1 hr is too short at low temperature. Alternatively, you can incubate with the primary antibody for 1-2 hrs at room temperature.

Have you tried to dilute the secondary antibody further down to see if the non-specific staining is getting fainter? My personal experience is that dilution 1:200 or 1:100 of a secondary antibody may be high.

I would advise you please to take a look at the on-line Protocol, you may find some useful information:

http://www.abcam.com/index.html?pageconfig=resource&rid=10381

http://www.abcam.com/assets/pdf/protocols/Immunocytochemistry%20(ICC)%20protocol.pdf

I hope this will be useful for you.

DESCRIPTION OF THE PROBLEM nonspecific stainning all over the cell by immunoflurescence.

SAMPLE Human breast cells growm on coverslip.

PRIMARY ANTIBODY ab4 and ab17298

DETECTION METHOD immuno flourescence

POSITIVE AND NEGATIVE CONTROLS USED No first antibody control is clean.

ANTIBODY STORAGE CONDITIONS in the fridge.Only purchased 2 weeks ago.

SAMPLE PREPARATION Fiexed in 4% para and permiabalised using 0.25% triton 100 in pbs.

TRANSFER AND BLOCKING CONDITIONS PBS with 10% fcs

SECONDARY ANTIBODY alexa fluor 488

DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I have diluted the primary and the secodary many folds.No Joy

ADDITIONAL NOTES I have spoken to a person at your section ,followed all the recommendations.Still no joy.Would like a reference wher your antibodies being used.Ieab17298 and ab 4

ANSWER:

Thank you for getting back to me with details of your technical approach.

Further to our telephone conversation I have searched the literature and I have determined that Calreticulin antibody - ER Marker (ab4) was used in the following publication; Luo X et al. C1q-calreticulin induced oxidative neurotoxicity: relevance for the neuropathogenesis of Alzheimer's disease. J Neuroimmunol 135:62-71 (2003). PubMed: 12576225. In this publication it details the approach adopted for the staining of rat cortical neurons. I consider this a good approach for the use of this antibody. In the methodology they detail;

2.5. Immunofluorescence staining of CaR

To visualize CaR, RCN were cultured in 24-well plates and then fixed with 2% paraformaldehyde and 120 mM sucrose in phosphate-buffered saline (PBS) for 10 min at room temperature. After quenching paraformaldehyde with 0.1 M glycine in PBS, the samples were blocked with 50% goat serum, 1% bovine serum albumin (BSA) in PBS (blocking buffer) for 40 min. Cells were subsequently incubated with a rabbit polyclonal antibody against CaR in blocking buffer for 40 min at room temperature. After several washes with PBS, cells were incubated with Alexa-594 (red fluorescence)-conjugated goat anti-rabbit IgG (1:50 dilution, preabsorbed IgG for multiple staining, Molecular Probes) for 30 min. After washes with PBS, cells were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and coverslip.

Whilst the approach that you have adopted is similar with respect to the the blocking and permeabilisation steps I consider the duration of fixation that you are adopting too long. As previously discussed with my colleague I would like to recommend a fixation period of 10-15 minutes following the approach above.

Unfortunately we do not have any publications that have applied ERAB antibody - Mitochondrial Marker (ab17298). However, once again I would like to suggest that you try applying this antibody following milder fixation duration of 2% for 10 minutes.

Should this not improve the results that you have been obtaining please get back in touch with me.

Hi!

I tried to perform Western blots with your antibody, but I did not have any results. The samples were as follows: rat bone extract, mouse muscle extract and bovine recombinant calreticulin. I used the method according to Towbin with the dilution of 1:1000, and did not get any bands. Do you have any idea why?

ANSWER:

We are not familiar with the method according to Towbin. 1. Can you describe this method in detail? 2. Did the antibody arrive in good condition? at a cold temperature? 3. Did you run any negative controls?

If the recombinant protein did not work this is quite suprising as it has been tested on endogenous bovine tissue (NBL-1 bovin renal epithelial cell line).

Dear Sirs, have you any experience with the calreticulin antibody concerning staining of ER in MDCK- and/or COS7- and/or HEK 293 cells? Which protocol/dilution of the antobody do you suggest?

ANSWER:

We do not have any specific experience with the staining of these cell lines. It has been tested successfully in human, bovine, rat, hamster, and mouse, but other species, such as canine, are untested. As for dilutions, 1:100~1:250 can be used according to the instruction in Heal and McGivan. Biochem. J. 329:389-394, 1998, which also describes the production of this antibody.