Anti-GST antibody [3G10/1B3] (ab92)

Hi,   I am wondering what would you recommend me to use as a loading control for human serum sample? Your website only showed what you have available, but I want to know what I can use?  I have been your beta-actin for most of my experiment and it works great!

ANSWER:

I am very glad to hear that our anti-Beta Actin loading control has worked so well for you.

In regards to a loading control for your current needs, Beta Actin would only be present in serum if shearing of cells had occurred during collection so this would not make a viable control. I have come across data stating that tranferrin and alpha anti-Trypsin are usually expressed at levels ˜2.5mg/ml and ˜1.3mg/ml respectively. However, as these may vary in samples, it may be best to add a purified peptide such a GST to your sample and stain against that.

We do offer antibodies against Transferrin, alpha anti-Trypsin and GST as well as purified GST peptides.

GST (ab70456):http://www.abcam.com/GST-protein-ab70456.html

anti-GST antibody (ab92):http://www.abcam.com/GST-antibody-3G10-1B3-ab92.html

anti-alpha-anti-Trypsin antibody (ab7633):http://www.abcam.com/alpha-1-Antitrypsin-antibody-ab7633.html

anti-Tranferrin antibody (ab1223):http://www.abcam.com/Transferrin-antibody-ab1223.html



I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

BATCH NUMBER 10PX402 ORDER NUMBER 598807

DESCRIPTION OF THE PROBLEM smudged/fuzzy band(s)?

SAMPLE Mouse liver samples

PRIMARY ANTIBODY Mouse anti-GST (abcam #ab92) was used at a dilution of 1:1000 (3uL in 3mL of PBS/0.05% Tween20) This incubated with rotation for 2 hour at RT. The membrane was given a quick wash and then washed 4 x 5 min. with the PBS/Tween.

DETECTION METHOD Western Blue Stabalized Substrate for AP (Promega #S3841)

POSITIVE AND NEGATIVE CONTROLS USED nothing

ANTIBODY STORAGE CONDITIONS Aliquoted with current tube stored at -20 degC and the rest stored at -80 degC.

SAMPLE PREPARATION Livers were sonicated in NETT buffer pH 7.5 with Protease Inhibitor Cocktail Set III (EMD Biosciences #539134), adding the PI according to the weight of the livers. They were then diluted 1:10 with additional NETT buffer and 1:100 PI. It is quantitated and 20-50ug are used per lane. A 4x loading buffer is adding and the sample is heated at 95 degC for 5-10 min.

AMOUNT OF PROTEIN LOADED The most recent was ~50 ug because we were having problems detecting lower levels of protein.

ELECTROPHORESIS/GEL CONDITIONS 12% Polyacrylamide gel with stacking gel. These were run in a Tris-Glycine-SDS electrophoresis buffer for ~4 hours. It was run at 60V through the stacking gel and then 120-150V through the running gel.

TRANSFER AND BLOCKING CONDITIONS They were transferred using a Tris-Glycine-Methanol buffer for 1 hour at 100V. They were then blocked overnight at 4 degC with 5% dry non-fat milk in PBS/0.2% Tween20/0.02%Na-azide. The membranes were subsequently rinsed with PBS/0.05%Tween20 before the primary antibody.

SECONDARY ANTIBODY Goat anti-mouse AP (Promega #s3721) was used at a dilution of 1:7500 in PBS/0.05%Tween20). This incubated with rotation for 1 hour at RT (this Ab is known to work with other primary antibodies). The membrane was quickly washed and then 4 x 5 min. washes with the PBS/Tween.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? The first attempt was a dilution of the primary antibody at 1:500, the second was 1:2000, and this 3rd was 1:1000. The blot is always fuzzy. The primary has been left overnight at room temperature, overnight at 4 degC and 2 hours at room temp, respectively.

ADDITIONAL NOTES I'm attaching an image of the blot. The bottom half is the portion stained for GST. The band(s)? is in the correct location, but it is so unclear that it is hard to tell if there are multiple bands and also difficult to quantitate because of this. The top section is not the top of this blot, but the top was stained with a different antibody and it had bands similar to this... so it is possible to stain with other antibodies and get nice bands.

ANSWER:

I'm sorry to hear you are experiencing problems with ab92.

I have been able to source the images from the laboratory and have added them to the datasheet, interestingly the band we get with a HeLa whole cell lysate is also large ("fat") in a similar way to your blot so it may be that the protein's migration properties give this pattern.

I enclose below other possible reasons for the problem which accentuate the band fuzziness:

1) too little Tween20 in the buffer: I would recommend adding more Tween20 to the antibody dilution buffer/washing buffer (0.1%v/v) and using a Tris based buffer rather than PBS as Tris gives sharper bands.

2) the percentage of SDS/methanol in the migration buffer and transfer buffer respectively can be a problem (SDS percentage should be around 1%w/v and should be fresh SDS, methanol percentage should be 20%v/v). The stacking gel composition can give problems with small proteins and resolution of small proteins often does not give as sharp a band as large proteins.

3) the blocking buffer can also sometimes give problems to the antibody and prevent it from binding correctly. I suggest trying 1hr milk 5% and incubation of the antibody in TBST only and also try 5%BSA the same way.

3) the lysis buffer (NETT buffer) may not be the most suitable extraction buffer for the protein. We do not have experience of this buffer and I would recommend trying a 20 mM Tris-HCl, pH 7.5 1 mM EGTA buffer or a NP40/RIPA buffer (50mM Tris HCl pH 8, 150 mM NaCl, 1 % NP-40, 0.5 % sodium Deoxycholate, 0.1 % SDS).

4) Finally, we have had good results with even more dilute antibody (0.5ug/ml equivalent to 1:2000) therefore you may find that by diluting the antibody more (and with the change in blocking buffer) and incubating at 4C overnight as you have previously done this will improve the signal (even if you need to expose the blot longer).

I hope these suggestions will help you, please do not hesitate to contact me if you require further assistance,

Thanks for your fast reply. I wonder if you would like to reward me another free sample of this product or a catalog of your company? That would be more appreciated.

ANSWER:

Thanks again for your email. We do not routinely offer free or trial sized samples for testing purposes. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement.

Should you decide to test an antibody in an application for which we do not have any information, please let us know how you get on and in return we will award you 50 points with the Abcam Loyalty Scheme which can be redeemed on a number of rewards.

Regarding a catalogue, we don't have a traditional "paper" catalogue as all of our items are listed online on our website www.abcam.com

Please let me know if you have any additional questions.

on the datasheet, it's said this antibody was raise by targetting 1-425aa of human GST. I didn't find any HUMAN GST had 425aa, normally they were 200aa. I need to know the exact seqence of the antigen which you used to raise this antibody.

ANSWER:

Thank you for enquiry. I have checked with the originator of this antibody and there was a mistake regarding the immunogen on the datasheet.

This antibody was generated as a bi-product during a fusion. The original immunogen was a GST fusion protein, with the total fusion protein being 425 amino acids. The actual GST portion is 232 amino acids: MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLIEGRGIPGNSS

Unfortunately I am unable to share what the protein fragment was that was fused to GST during the generation of this particular clone due to confidentiality reasons. I can say is that it was a small fragment of a very high molecular weight protein.

I have updated the datasheet to reflect this information and apologize for the inconvenience. If you have any additional questions, please contact us again.