Bovine Heart Mitochondria (ab110338)

Product ab110338 contains whole mitochondria in iso-osmotic buffer. There is no need to measure protein concentration of this product; the product is supplied at 5.5 mg/mL. Simply adjust the protein concentration with PBS to 5.3 mg/mL and add detergent according to the kit’s protocol.
If you wish to measure the protein concentration of ab110338, dilute an aliquot according the assay range (100-200x using Pierce Micro BCA) in 0.25% SDS, this will solubilise the mitochondrial membranes. Do not use this aliquot to perform the assay in kit ab109902. There is no need to homogenize the sample for BCA or for the kit’s assay.

Le principe de base que nous utilisons pour la préparation d’ab110338 est le même que dans le kit https://www.abcam.com/mitochondria-isolation-kit-for-tissue-ab110168.html.

La seule différence c’est que nous adaptons le protocole pour travailler avec de plus gros volumes (un cœur bovin entier).

- Toute la graisse et le tissu cartilagineux dans le cœur est enlevé et jeté (graisse péricardique, vannes). Cela laisse la plupart du temps les ventricules.
- Le tissu est haché dans un mélangeur à faible vitesse (au lieu d'utiliser un homogénéisateur Dounce)
- Après la première et la deuxième centrifugation, nous récupérons le surnageant et nous le filtrons à travers une étamine.
- Après cela, nous effectuons trois centrifugations à 12 000 g.

The ration among complexes varies from tissue to tissue. We did not know the exact number but when we validated the expression of different complexes using this product, we did observed variation across tissues.

J’ai contacté le laboratoire concernant votre question pour ab110338. Ab110338 ne contient pas de détergent. Ce lysat devrait contenir des enzymes actives et donc pourrait être utilisé pour des tests enzymatiques. Cependant, avant d’utiliser ab110338, le lysat doit être solubilisé. Nous recommandons le détergent ab109857 (10% Lauryl Maltoside Solution).

Thank you for your enquiry and interest in our product.

The concentration comes at 5.5 mg/mL (360uL). It is done in this way so that our customers can add 40uL of 10% lauryl maltoside for a final volume of 400uL at a concentration of 5 mg/mL in preparation for IP experiments.

If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for your enquiry and interest in our product.

I can confirm that the mitochondria preparation is whole mitochondria (intact) but because the mitochondria is frozen it is not coupled.

By this I mean you are not able to perform respiration experiments with either the clark electrode or the seahorse instrument.

The preparation is made from fresh bovine tissue so it is of fairly good quality. However, it may contain traces of other organelles such as peroxisomes, ER or cytoplasmic proteins.

If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for your reply.

The WB looks great now - with the bright band at around 37 kDa. The band at the higher MW is very faint and seems to be specific to your sample or sample preparation as the mitochondrial control lysate does not have this band.

I also looked at the images you had sent earlier. The blots from 2010 also showed a weak band at the higher MW. If the same samples were used, than that would confirm that this band is indeed related to your samples. The bovine mito samples do not show such band.

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

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Oh, I see.

The bovine heart mito lysate was prepared with iso-osmotic buffer, thus without detergent - so that it can be used for various experiments not just WB.

Additional information as per datasheet:
2 mg of bovine heart mitochondria in iso-osmotic buffer. This sample can act as a positive control in immunocapture applications and Blue Native PAGE.

1 mg solubilized mitochondria for Immunocapture experiments. For the Immunocapture of OXPHOS complexes, it is recommended that 180 µl of the mitochondrial membranes supplied be solubilized by the addition of 20 µl of 10% lauryl maltoside. Insoluble material is removed by centrifugation at 72,000g before immunocapture of complexes from the supernatant. For more detail see Abcam's suggested protocols.


Thus, the preparation of the lysate in this way, works to obtain the expected band at 37 kDa. It seems therefore that the use of 8M urea has a different affect on your samples which might cause it to run at the higher MW.

I would suggest - in light of your results - to try lysing your cells either with an iso-osmotic buffer (mechanical lysis) or an SDS-based buffer. That should help with obtaining the band at the expected MW.

I hope this is of help. Please let me know if you have more questions.

Thank you for your reply.

The lab let me know in the meantime that they think you have used too much bovine heart mito lysate. Usually, the loading amount for the bovine heart mito lysate should be at least one tenth of the cell lysates. The band shows up at the correct MW, the additional smear above could be due to overloading the well.

Also, the lab agrees that you will get better result if you use SDS-loading buffer instead of 8M urea for denaturing.

Our WB protocol contains the information about the SDS loading buffer:

https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A6

"The standard loading buffer is called 2X Laemmli buffer, first described in Nature, 1970 Aug 15;227(5259):680-5. It can also be made at 4X and 6X strength to minimize dilution of the samples. The 2X is to be mixed in a 1:1 ratio with the sample.

Laemmli 2X buffer

4% SDS
10% 2-mercaptoehtanol
20% glycerol
0.004% bromophenol blue
0.125 M Tris HCl

Check the pH and bring it to pH 6.8.

When SDS is used with proteins, all of the proteins become negatively charged by their attachment to the SDS anions. SDS denatures proteins by “wrapping around” the polypeptide backbone. SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - i.e., the denatured polypeptides become “rods” of negative charge clouds with equal charge or charge densities per unit length.

In denaturing SDS-PAGE separations, therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. SDS grade is of utmost importance: a protein stained background along individual gel tracts with indistinct or slightly distinct protein bands are indicative of old or poor quality SDS. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size by using ß-mercaptoethanol or dithiothreitol (DTT).

Glycerol is added to the loading buffer to increase the density of the sample to be loaded and hence maintain the sample at the bottom of the well, restricting overflow and uneven gel loading."



The bovine lysate does not show a band around 85-90 kDa, thus indicating that the kit and antibodies work as expected. It might be that the way your samples were prepared differs from how the bovine lysate was prepared, thus resulting in the difference in MW of the Cox-1 bands.

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

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Thank you for your email.

Was the bovine heart mitochondrial lysate also prepared with 8M urea?

I am not sure if this is what the lab recommends for denaturing. I think regular loading buffer with 1% SDS would be recommended (Laemmli buffer).

I will check with them, but in the meantime please let me know as well.

Thank you!