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General Materials

Opti-Protein Marker

Cat. No.

G252

Unit

500 μl/100 loads

Price

$113.00

Cat. No.	G252
Name	Opti-Protein Marker
Unit	500 μl/100 loads
Category	DNA & Protein Ladders
Description	Opti-Protein Marker/Ladder consists of 11 proteins that resolve into sharp, tight bands in the range of 10-175 kDa. This unique protein ladder allows you to monitor protein separation during electrophoresis, estimate molecular weight of the protein of interest, and evaluate western blot transfer efficiency. It also includes ~10, ~40 and ~90 kDa reference bands coupled with blue dye that allow easy tracking and identification. Number of Bands: 11 Protein Range: 10kDa-175kDa Suggested Load: Mix gently by pipetting up and down a few time, then directly load 5 μl into well of gel.
Application	• Monitoring protein migration during SDS-PAGE • Monitoring efficiency of protein transfer onto PVDF or nitrocellulose for Western Blotting • Sizing of proteins on SDS-PAGE and Western Blots
Storage Condition	Store @ 4°C for 3 months or -20°C for 24 months.
Material Citation	If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. G252

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Supporting Protocol

Protein Markers

	How many times can a tube be freeze/thawed? Can secondary antibody detect the ladder bands?
	It could be freeze/thawed at least 100 times. Secondary antibody cannot detect the ladder.

The marker should include proteins with defined molecular weights. However, the 3 images show different kDa numbers in the MOPS and MES buffer compared to the Tris Glycin Buffer. Why?

The standards in the protein markers have different forms in different gel conditions. With the change in structure, the migration speed is altered. The markers are then re-standardized in each condition against a known protein size. Yes, the protein base for each marker remains the same size. However, the protein is conjugated to a dye in our prestained ladder. Thus, the protein+dye complex migrates differently than an unmarked protein of the same size. Our figures has been calibrated to match the size of unstained protein with the protein+dye complex at the same migration distance. This is because customers will be using unstained proteins with our prestained ladder. To summarize, the kDa of bands in prestained protein markers do NOT mean the real molecular weight of protein bands (the stained protein). The kDa means the molecular weight of unstained proteins.

Why I do have high background?

Blocking of non-specific binding might be absent or insufficient. Increase the blocking incubation period and consider changing blocking agent. abm recommends blocking for 30 minutes with 5% non-fat dry milk, 3% BSA, or normal serum. These can be included in the antibody buffers as well. The primary antibody concentration may be too high. Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but targeted binding is best). Incubation temperature may be too high. Incubate blot at 4°C. The secondary antibody may be binding non-specifically or reacting with the blocking reagent. Run a secondary control without primary antibody. Cross-reaction between blocking agent and primary or secondary. Add a mild detergent such as Tween20 to the incubation and washing buffer. (phospho-specific protein) Milk contains casein which is a phosphoprotein; this is why it causes high background because the phospho-specific antibody detects the casein present in the milk. Use BSA as a blocking reagent instead of milk. Washing of unbound antibodies may be insufficient. Increase the number of washes. Your choice of membrane may give high background. Nitrocellulose membrane is considered to give less background than PVDF. The membrane has dried out. Care should be taken to prevent the membrane from drying out during incubation.

Why do I have multiple bands?

1. Cell lines that have been frequently passaged gradually accumulate differences in their protein expression profiles. - Go back to the original non-passaged cell line and run the current and original cell line samples in parallel. 2. The protein sample has multiple modified forms in vivo such as acetylation, methylation, myristylation, phosphorylation, glycosylation etc. - Examine the literature and use an agent to dephosphorylate, de-glycosylate, etc. the protein to bring it to the correct size. 3. The target in your protein sample has been digested (more likely if the bands are of lower molecular weight). - Make sure that you incorporate sufficient protease inhibitors in your sample buffer. 4. Unreported novel proteins or different splice variants that share similar epitopes and could possibly be from the same protein family are being detected. - Check the literature for other reports and also perform a BLAST search; Use the cell line or tissue reported on the datasheet. 5. Primary antibody concentration is too high - at high concentration multiple bands are often seen. Try decreasing the antibody concentration and/or the incubation period. 6. Secondary antibody concentration is too high - at high concentration secondaries will bind non-specifically. - Try decreasing the concentration. Run a secondary antibody control (without the primary). 7. The antibody has not been purified. - Try to use affinity purified antibody. This will often remove non-specific bands. 8. The bands may be non-specific. - Where possible use blocking peptides to differentiate between specific and non-specific bands. Only specific bands should be blocked (and thus disappear). 9. The protein target may form multimers. - Try boiling in SDS-Page for 10 minutes rather than 5 minutes to disrupt multimers.

	Why are there uneven white “spots” on the blot?
	Air bubbles were trapped against the membrane during transfer or the antibody is not evenly spread on the membrane. Make sure you remove bubbles when preparing the gel for transfer. Incubate antibodies under agitation.

	Why do I have black dots on the blot?
	The antibodies are binding to the blocking agent. Filter the blocking agent.

	White bands on a black blot (negative of expected blot)?
	Too much primary and/or too much secondary antibody. Dilute the antibodies more.

	MW marker lane is black?
	The antibody is reacting with the MW marker. Add a blank lane between the MW marker and the first sample lane.

	The band of interest is very low/high on the blot?
	It is due to separation is not efficient. Change the gel percentage: a higher percentage for small protein, lower percentage for large proteins.

	Smile effect of the bands?
	Two possibilities: 1. Migration was too fast. 2. Migration was too hot (changing the pH and altering the migration). Slow down the migration or run the gel in the cold room or on ice.

	Uneven band size in lanes probed for the same protein?
	Gel has set too quickly while casting and the acrylamide percentage is not even along the lanes. Review the recipe of the gel and the addition of TEMED to the gels, add a little 0.1% SDS in water to the top of the migrating gel while it sets to stop it from drying.

	Uneven staining of the gel?
	Two possibilities: 1. Contamination from bacteria 2. Not enough antibody 1. Keep antibodies at 4°C and use fresh buffers covers the gel. 2. Make sure the membrane is covered with the antibody/ incubate under agitation.

	Is it pre-stain or un-stain?
	All the OptiProtein marker are pre-stained and ready to load directly on the gel.

I do not see any bands on my blot. Are there any suggestions?

The best possible control for the antibody is to transfect 293 cells with your gene of interest, produce lysate from these cells, and then use them for western blot. For other lysates from cells that do express the protein of interest naturally, the expression level may not be strong enough for detection and it is best to work with 293 cells first to prove if the antibody is functional in your hands. Also, removing all detergent and incubating the primary antibody overnight will help produce stronger band signals.

	When you say Tris-Glycine 15%, are you referring to the gel or the buffer?
	Tris-Glycine 15% refers to the gel conditions.

	How many micrograms of protein are in each band of the marker?
	We have about 0.1-0.2mg per protein band for each 500ul vial of the prestained protein ladder.

	Are Opti-Protein Markers visible under infrared or UV light?
	No, this will require staining with other dyes.

	Is there any EDTA present in the marker solution for proper running of Phos-Tag gels?
	Yes, EDTA is present at a concentration of 1mM.

	Should I heat it before I load?
	Recommendations for Loading: 1. Thaw the ladder either at room temperature or at 37-40°C for a few minutes to dissolve precipitated solids. Do not boil. 2. Mix thoroughly to ensure the solution is homogeneous. 3. Load the following volumes of the ladder on SDS-polyacrylamide gel: - 5 μl per well for mini-gels, 2.5 μl per well for blots - 10 μl per well for large gels, 5 μl per well for blots

	Is this protein marker adapted for use in native gels?
	No, this protein marker is adapted for use in SDS-PAGE. Proteins do not migrate based on molecular weight, but rather based on charge to mass ratio in native gels.

	Do the protein markers work against native gels?
	Our protein markers can run though a native gel. However, all our protein markers contain denatured proteins and thus, cannot be equally compared to native proteins in the native gel setting.

Li, HL et al. "Overexpression of MACC1 and the association with hepatocyte growth factor/c-Met in epithelial ovarian cancer" Oncology Letters 5:1989-1996 (2015). DOI: 10.3892/ol.2015.2984. Application: Ladder.
Sharma, V et al. "Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation" eLIFE 5 (e11414): (2016). DOI: 10.7554/eLife.11414. Application: Western Blot.
Takci, HAM et al. "Extracellular Pectinase Production and Purification from a Newly Isolated Bacillus subtilis Strain" International Journal of Food Properties 19 (11):2443-2450 (2016). DOI: 10.1080/10942912.2015.1123270. Application: SDS-PAGE.
Sharma, V et al. "Registered Report: Diverse somatic mutation patterns and pathway alterations in human cancers" eLIFE 5 (e11566): (2016). DOI: 10.7554/eLife.11566. Application: Western Blot.

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